Methods and compositions for improving detection and/or capture of a target entity

ABSTRACT

Methods, compositions, kits and systems for detecting and/or capturing a target entity in a sample. In particular, the methods, compositions and kits described herein can be used for pre-treatment of target-binding agents with a blocking agent to reduce non-target binding in a complex matrix (e.g., blood). Methods and compositions for detecting and/or capturing a microbe in a test sample, including bodily fluids such as blood and tissues of a subject, food, water, and environmental surfaces are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 14/766,575, filed Aug. 7, 2015, which is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2014/028683 filed Mar. 14, 2014, which designatesthe U.S., and which claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 61/788,570 filed Mar. 15, 2013, the contentsof each of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 6, 2015 isnamed 20150807_Sequence_Listing_002806-076402-US.txt and is 18,135 bytesin size.

TECHNICAL FIELD

Described herein relates generally to methods, compositions, and kitsfor detecting and/or capturing a target entity in a sample. In someembodiments, methods and compositions for detecting and/or capturing amicrobe in a test sample, including bodily fluids such as blood andtissues of a subject, food, water, and environmental surfaces are alsoprovided herein.

BACKGROUND

Sepsis is a major cause of morbidity and mortality in humans and otheranimals. In the United States, sepsis is the second leading cause ofdeath in intensive care units among patients with non-traumaticillnesses. It is also the leading cause of death in young livestock,affecting 7.5-29% of neonatal calves, and is a common medical problem inneonatal foals. Despite the major advances of the past several decadesin the treatment of serious infections, the incidence and mortality dueto sepsis continues to rise.

Sepsis results from the systemic invasion of microorganisms into bloodand can present two distinct problems. First, the growth of themicroorganisms can directly damage tissues, organs, and vascularfunction. Second, toxic components of the microorganisms can lead torapid systemic inflammatory responses that can quickly damage vitalorgans and lead to circulatory collapse (i.e., septic shock) and, oftentimes, death.

Sepsis is a systemic reaction defined by the American College of ChestPhysicians and the Society of Critical Care Medicine by a systemicinflammatory response (SIRS) in response to a confirmed infectiousprocess. SIRS is defined by the presence of two or more of thefollowing: altered body temperature (<36° C. or >38° C.), tachycardia(heart rate>90/min), tachypnea (respiratory rate>20/min) or hypocapnia(P_(a)CO₂ less than 4.3 kPa), leucopenia (white blood cells (WBCs) <4000cells/mm³ or leucocytosis (>12000 WBC/mm³) or >10% band forms. Theconfirmation of the infectious process is confirmed by microbiologicalmeans (stain, culture, antigenemia or antigenuria, nucleic aciddetection) or pathognomonic signs of infection obtained by imaging orclinical examination. The infection can affect any organ system, but themore severe cases present as septicemia (i.e., organisms, theirmetabolic end-products or toxins in the blood stream), bacteremia (i.e.,bacteria in the blood), toxemia (i.e., toxins in the blood), andendotoxemia (i.e., endotoxin in the blood). Sepsis can also result fromfungemia (i.e., fungi in the blood), viremia (i.e., viruses or virusparticles in the blood), and parasitemia (i.e., helminthic or protozoanparasites in the blood). Thus, septicemia and septic shock (acutecirculatory failure resulting from septicemia often associated withmultiple organ failure and a high mortality rate) may be caused byvarious microorganisms.

There are three major types of sepsis characterized by the type ofinfecting organism. For example, gram-negative sepsis is the mostfrequently isolated (with a case fatality rate of about 35%). Themajority of these infections are caused by Escherichia coli, Klebsiellapneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such asthe Staphylococci and Streptococci are the second major cause of sepsis.The third major group includes fungi, with fungal infections causing arelatively small percentage of sepsis cases, but with a high mortalityrate; these types of infections also have a higher incidence inimmunocompromised patients.

Some of these infections can be acquired in a hospital setting and canresult from certain types of surgery (e.g., abdominal procedures),immune suppression due to cancer or transplantation therapy, immunedeficiency diseases, and exposure through intravenous catheters. Sepsisis also commonly caused by trauma, difficult newborn deliveries, andintestinal torsion (especially in dogs and horses). Infections in thelungs (pneumonia), bladder and kidneys (urinary tract infections), skin(cellulitis), abdomen (such as appendicitis), bone (osteomyeltitis) andjoints (arthritis) and other areas (such as meningitis) can spread andalso lead to sepsis. In some circumstances, ingestion ofmicrobe-contaminated water, fluid or food, or contact withmicrobe-covered environmental surfaces can cause infections that lead tosepsis, and infection with food-borne and water-borne pathogens such asShigella spp, or certain serotypes of Escherichichia coli (such as O157H7), Salmonella spp including Salmonella enterica serovar typhi orListeria monocytogenes can also lead to sepsis.

Many patients with septicemia or suspected septicemia exhibit a rapiddecline over a 24-48 hour period. It has been reported that patientswith septic shock require adapted treatment in less than 6 hours inorder to benefit from antimicrobial therapy. Thus, rapid and reliablediagnostic and treatment methods are essential for effective patientcare. Unfortunately, a confirmed diagnosis as to the type of infection,e.g., sepsis, traditionally requires microbiological analysis involvinginoculation of blood cultures, incubation for 18-24 hours, plating thecausative microorganism on solid media, another incubation period, andfinal identification 1-2 days later. Even with immediate and aggressivetreatment, some patients can develop multiple organ dysfunction syndromeand eventually death. Hence, there remains a need for improvedtechniques for diagnosis of patients with infectious diseases,blood-borne infections, sepsis, or systemic inflammatory responsesyndrome. In addition, the ability to detect infectious pathogens infood, water, and/or environmental surfaces with improved specificity andthus decreased incidence of false positives would help providingappropriate and necessary treatments to patients who are in need andthus reducing healthcare cost.

SUMMARY

Embodiments of various aspects described herein are, at least in part,based on discovery of pre-treating a target-binding agent with anappropriate concentration of an intermediate-affinity ligand for thetarget-binding agent, prior to contacting a sample with thetarget-binding agent, so as to reduce non-target binding during captureof a target entity in the sample (e.g., blood). Unlike a typicalblocking agent commonly used to saturate unoccupied binding sites on aninterfering agent (non-target material), the inventors have discoveredthat pre-treating a microbe-binding agent comprising a mannan-bindingdomain (e.g., FcMBL, which is a fusion protein or peptide comprising acarbohydrate recognition domain of a mannan-binding lectin and a Fcportion of an immunoglobulin) with an intermediate-affinity blockingagent (e.g., glucose) can not only improve binding specificity and/orsensitivity of the microbe-binding agent for microbes and/or fragmentsthereof (target entity), but can also reduce false positives resultingfrom non-target binding (e.g., haemocyte binding). In one embodiment,glucose is selected as one of the blocking agents for use in the FcMBLsystem to detect and/or capture microbes or fragments thereof, partlybecause glucose can prevent non-target or interfering agents such ashaemocytes from binding to FcMBL, while permitting a target entity suchas microbes and/or fragments thereof to displace glucose that is boundto FcMBL.

The concept of pre-treatment of a target-binding agent with a blockingagent, where the blocking agent is selected based on relative bindingaffinities of the blocking agent, a target entity and an interferingagent, respectively, for the target-binding agent, can be extended toany detection/capture processes, assays, systems and/or platforms inwhich binding interaction between the target entity and thetarget-binding agent is involved in a sample comprising at least oneinterfering agent. The blocking agent used in these detection/captureprocesses, assays, systems and/or platforms can be selected to have aneffective binding affinity for the target binding agent that is betweenan effective binding affinity of a target entity for the target-bindingagent and the effective binding affinity of an interfering agent for thetarget-binding agent, so that the target entity, but not the interferingagent, can displace the blocking agent that is bound to thetarget-binding agent, and thus be captured on the target-binding agent.Accordingly, embodiments of various aspects described herein relate tomethods, compositions and kits for detecting or capturing at least onetarget entity.

In one aspect, provided herein relates to methods of detecting orcapturing at least one target entity. The method comprises contacting asample with a composition comprising a target-binding agent and ablocking agent, wherein the blocking agent is selected for reducing thebinding of at least one interfering agent present in the sample to thetarget-binding agent, while permitting a first target entity, if presentin the sample, to (a) displace the blocking agent bound to thetarget-binding agent, or to (b) bind to the target-binding agent withoutthe blocking agent bound thereto. In some embodiments, the blockingagent is bound to the target-binding agent within the composition, priorto contacting the sample with the composition.

In some embodiments of this aspect and other aspects described herein,the effective binding affinity of the blocking agent for thetarget-binding agent can be selected for increasing apparent specificityof the target-binding agent to the first target entity in the sample, ascompared to the apparent specificity in the absence of the blockingagent. For example, the apparent specificity of the target-binding agentto the first target entity can be increased by reducing the binding ofsaid at least one interfering agent to the target-binding agent, whichcan in turn increase the availability of binding sites on thetarget-binding agent for the target entity. While the binding of said atleast one interfering agent to the target-binding agent is typicallyreduced by blocking the unoccupied binding sites on the interferingagent, the inventors instead pre-block or mask the binding sites on thetarget-binding agent with a blocking agent such that the interferingagent cannot bind to the target-binding agent, but the target entity isable to displace the blocking agent due to the target entity's higheraffinity for the target-binding agent than that of the blocking agent.Thus, the target entity is bound to a sub-population of thetarget-binding agent, while a sub-population of the target-binding agentcan still comprise the blocking agent bound thereto. The target entitycan then be detected, for example, with a detection agent. In someembodiments, the blocking agent that is bound to the target-bindingagent cannot bind to a detection agent and thus cannot interfere withdetection of the target-binding agent bound with a target entity. Inother embodiments, a blocking agent that is already bound to thetarget-binding agent can still bind to a detection agent. In theseembodiments, the blocking agent can be treated, prior to addition of adetection gent, to mask its spare binding sites such that the blockingagent becomes unable to bind to the detection agent.

Accordingly, in some embodiments, the blocking agent can be selectedbased on its effective binding affinity relative to effective bindingaffinities of the first target entity and said at least one interferingagent, respectively, for the target-binding agent. In some embodiments,the blocking agent can be selected to have an effective binding affinityfor the target-binding agent that is lower than an effective bindingaffinity of the first target entity for the target-binding agent; andthe blocking agent is further selected to have the effective bindingaffinity for the target-binding agent that is higher than an effectivebinding affinity of at least one interfering agent present in the samplefor the target-binding agent.

In order to permit the first target entity to displace the blockingagent bound to the target-binding agent, the effective binding affinityof the blocking agent for the target-binding agent is desired to belower than the effective binding affinity of the first target entity forthe target-binding agent. In some embodiments of this aspect and otheraspects described herein, the effective binding affinity of the blockingagent for the target-binding agent can be lower than the effectivebinding affinity of the first target entity for the target-binding agentby at least about 10% or more, including, e.g., at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or more.

In order to prevent at least one interfering agent from binding to thetarget-binding agent, the effective binding affinity of the blockingagent for the target-binding agent is desired to be higher than theeffective binding affinity of said at least one interfering agentpresent in the sample for the target-binding agent. In some embodimentsof this aspect and other aspects described herein, the effective bindingaffinity of the blocking agent for the target-binding agent can behigher than the effective binding affinity of said at least oneinterfering agent present in the sample for the target-binding agent byat least about 10% or more, including, e.g., at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or more.In some embodiments, the effective binding affinity of the blockingagent for the target-binding agent can be higher than the effectivebinding affinity of said at least one interfering agent present in thesample for the target-binding agent by at least about 1-fold or more,including, e.g., at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold or more.

In some embodiments of this aspect and other aspects described herein,the effective binding affinity of the blocking agent for thetarget-binding agent, as indicated by a dissociation constant for thebinding of the blocking agent to the target-binding agent, can rangefrom about 1 μM to about 100 mM, or about 10 μM to about 75 mM, or about5 mM to about 50 mM. In one embodiment, the effective binding affinityof the blocking agent for the target-binding agent, as indicated by adissociation constant for the binding of the blocking agent to thetarget-binding agent, can range from about 5 mM to about 50 mM. In someembodiments, the effective binding affinity of the blocking agent forthe target-binding agent, as indicated by a dissociation constant forthe binding of the blocking agent to the target-binding agent, can be ina nanomolar range. For example, in some embodiments, the effectivebinding affinity of the blocking agent for the target-binding agent, asindicated by a dissociation constant for the binding of the blockingagent to the target-binding agent, can range from about 1 nM to 10 μM,about 1 nM to about 1 μM, or about 10 nM to about 500 nM.

The blocking agent can be any molecule, compound, or agent that can bindto a target-binding agent with an effective binding affinity betweenthat of the target entity and the interfering agent, respectively, forthe target-binding agent. Depending on the binding properties of thetarget-binding agents, target entity, and/or interfering agents, theblocking agent can include, but not limited to, cells or fragmentsthereof, peptides, polypeptides, proteins, peptidomimetics, antibodies,antibody fragments (e.g., antigen binding fragments of antibodies),carbohydrate-binding protein, e.g., lectins, glycoproteins,glycoprotein-binding molecules, amino acids, carbohydrates (includingmono-, di-, tri- and poly-saccharides), lipids, steroids, hormones,lipid-binding molecules, cofactors, nucleosides, nucleotides, nucleicacids (e.g., DNA or RNA, analogues and derivatives of nucleic acids, oraptamers), peptide aptamers, peptidoglycan, lipopolysaccharide, smallmolecules, endotoxins (e.g., bacterial lipopolysaccharide), and anycombinations thereof. In some embodiments, the cells include, but arenot limited to, prokaryotes (e.g., microbes such as bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, and fungi), bloodcells, and any fragments thereof. In some embodiments where thetarget-binding agent is configured to recognize a carbohydrate pattern,e.g., for detection and/or capture of a microbe or a fragment thereof,the blocking agent can be a carbohydrate or a saccharide.

In some embodiments, the blocking agent can be a monomer, which has nofree binding site after binding to the target-binding agent. Thus, theblocking agent cannot subsequently bind to a detection agent, and thusprevent its interference with an assay used to detect a target entitybound to a target-binding agent. For example, a saccharide-basedmonomeric blocking agent can be a monosaccharide or modificationthereof.

In alternative embodiments, the blocking agent can be a multimer whichhas at least one free binding site after binding to the target-bindingagent. In these embodiments, the blocking agent can subsequently bind toa detection agent and thus interfere in an assay used to detect a targetentity bound to a target-binding agent. In these embodiments, theblocking agent can be treated, prior to addition of a detection agent,to mask all the free binding sites. In some embodiments, asaccharide-based multimeric blocking agent can be a disaccharide, anoligosaccharide, a polysaccharide, modifications thereof, or anycombinations thereof.

Examples of a saccharide-based blocking agent include, withoutlimitations, hexose (e.g., glucose), maltose, mannose, N-acetyl-muramicacid, amino sugars (e.g., galactosamine, glucosamine, sialic acid,N-acetylgludosamine), sulfosugars (e.g., sulfoquinovose), trehalose,cellobiose, lactose, lactulose, sucrose, fructo-oligosaccharides,cellulose, chitin, or any combinations thereof. In some embodiments, asaccharide-based blocking agent can be glucose, maltose,N-acetyl-muramic acid, or any combinations thereof. In one embodiment, asaccharide-based blocking agent can comprise glucose. In one embodiment,a saccharide-based blocking agent can comprise mannose.

The target entity can be any molecule, compound, or agent that can bedetected and/or captured by a target-binding agent. Non-limitingexamples of a target entity include, but are not limited to, cells orfragments thereof, peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., lectins, glycoproteins,glycoprotein-binding molecules, amino acids, carbohydrates (includingmono-, di-, tri- and poly-saccharides), lipids, steroids, hormones,lipid-binding molecules, cofactors, nucleosides, nucleotides, nucleicacids (e.g., DNA or RNA, analogues and derivatives of nucleic acids, oraptamers), peptide aptamers, peptidoglycan, lipopolysaccharide, smallmolecules, endotoxins (e.g., bacterial lipopolysaccharide), and anycombinations thereof. In some embodiments, the cells include, but arenot limited to, prokaryotes (e.g., microbes such as bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, and fungi), bloodcells, and any fragments thereof. In some embodiments, the target entitycan be a cell, e.g., but not limited to a microbe or a fragment thereof.

As described earlier, the effective binding affinity of the blockingagent for the target-binding agent is lower than the effective bindingaffinity of the target entity for the target-binding agent. Accordingly,in some embodiments of this aspect and other aspects described herein,the effective binding affinity of the first target entity for thetarget-binding agent, as indicated by a dissociation constant for thebinding of the first target entity to the target-binding agent, can beless than 100 mM, less than 75 mM, less than 50 mM, less than 25 mM,less than 10 mM, less than 5 mM, less than 1 mM, less than 0.5 mM, lessthan 0.1 mM, less than 10 μM, less than 1 μM, or less, provided that thedissociation constant for the binding of the blocking agent to thetarget-binding agent is smaller than higher than that for the binding ofthe first target entity to the target-binding agent. In someembodiments, the effective binding affinity of the first target entityfor the target-binding agent, as indicated by a dissociation constantfor the binding of the first target entity to the target-binding agent,can be less than 25 mM. In some embodiments, the effective bindingaffinity of the first target entity for the target-binding agent, asindicated by a dissociation constant for the binding of the first targetentity to the target-binding agent, can be in a nanomolar range. Forexample, in some embodiments, the effective binding affinity of thefirst target entity for the target-binding agent, as indicated by adissociation constant for the binding of the first target entity to thetarget-binding agent, can be less than 1 μM, less than 500 nM, less than100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5nM, less than 1 nM, less than 0.5 nM, or less.

The target-binding agent is an agent configured to detect and/or captureat least one target entity. The target-binding agent can be present inany form, including but not limited to a target-binding molecule, and/ora target-binding substrate (e.g., a target-binding molecule conjugatedto a solid substrate). In some embodiments, the target-binding agent cancomprise a target-binding molecule selected from the group consisting ofcells or fragments thereof, peptides, polypeptides, proteins,peptidomimetics, antibodies, antibody fragments (e.g., antigen bindingfragments of antibodies), carbohydrate-binding protein, e.g., lectins,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptide aptamers, peptidoglycan,lipopolysaccharide, small molecules, endotoxins (e.g., bacteriallipopolysaccharide), and any combinations thereof. In some embodiments,the target-binding agent can be configured to detect and/or capture atleast one microbe and/or a fragment thereof. Thus, in some embodiments,the target-binding agent can comprise a microbe-binding agent. By way ofexample only, a microbe-binding agent can comprise a lectin (e.g., aFcMBL molecule).

In some embodiments, the target-binding agent can be affixed to a solidsubstrate described herein. Non-limiting examples of a solid substrateinclude, but are not limited to, a nucleic acid scaffold, a proteinscaffold, a lipid scaffold, a dendrimer, microparticle or a microbead, ananotube, a microtiter plate, a medical apparatus or implant, amicrochip, a filtration device, a membrane, a diagnostic strip, adipstick, an extracorporeal device, a mixing element (e.g., a spiralmixer), a microscopic slide, a hollow fiber, a hollow fiber cartridge,and any combinations thereof.

An interfering agent is an agent present in a sample to be assayed,which undesirably binds to a target-binding agent and reduces theeffective binding affinity of the target-binding agent to thecorresponding target entity. In some embodiments where the samplecomprises a biological fluid, e.g., blood, at least one interferingagent can comprise a blood cell and/or a fragment thereof, e.g., a redblood cell (or an erythrocyte) and/or a fragment thereof. In someembodiments where the sample comprises a second target entity notintended to be captured or detected by the first target-binding agent,but by a second target-binding agent, the second target entity can beconsidered as an interfering agent with respect to the bindinginteraction between the first target-binding agent and the first targetentity. In some embodiments, said at least one interfering agent can bea non-specific binding molecule. In some embodiments, said at least oneinterfering agent can be a molecule for which the target-binding agenthas a binding specificity, but with a lower binding affinity than to thetarget entity.

As described herein, the effective binding affinity of said at least oneinterfering agent for the target-binding agent is lower than theeffective binding affinity of the blocking agent for the target-bindingagent. Accordingly, in some embodiments of this aspect and other aspectsdescribed herein, the effective binding affinity of said at least oneinterfering agent for the target-binding agent, as indicated by adissociation constant for the binding of the interfering agent to thetarget-binding agent, can be more than 5 μM, more than 10 μM, more than0.1 mM, more than 0.5 mM, more than 1 mM, more than 5 mM, more than 10mM, more than 25 mM, more than 50 mM, more than 75 mM, more than 100 mMor more, provided that the dissociation constant for the binding of theinterfering agent to the target-binding agent is larger than that forthe binding of the blocking agent to the target-binding agent. In someembodiments, the effective binding affinity of said at least oneinterfering agent for the target-binding agent, as indicated by adissociation constant for the binding of the interfering agent to thetarget-binding agent, can be more than 50 mM. In some embodiments, theeffective binding affinity of said at least one interfering agent forthe target-binding agent, as indicated by a dissociation constant forthe binding of said at least one interfering agent to the target-bindingagent, can be in a lower range, e.g., more than 500 nM, or more than 1μM, or higher.

In some embodiments, the method can further comprise exposing thetarget-binding agent to the blocking agent at a pre-determinedconcentration to form the composition comprising the target-bindingagent and the blocking agent bound thereto, prior to the contacting ofthe sample with the composition.

The blocking agent can be generally present in a sample at anyconcentration provided that its presence in the sample does notadversely affect the binding of the first target entity to thetarget-binding agent. In some embodiments, the blocking agent can bepresent in a pre-determined concentration that does not reduce thebinding of the first target entity to the target-binding agent by morethan 50%, no more than 40%, no more than 30% or less, as compared to thebinding in the absence of the blocking agent. In some embodiments, theconcentration ratio of the blocking agent to the target-binding agentcan range from about 100:1 to about 10,000:1, from about 250:1 to about7500:1, or from about 500:1 to about 5000:1. In some embodiments, theconcentration ratio of the blocking agent to the target-binding agentcan range from about 500:1 to about 5000:1. In some embodiments, theconcentration ratio of the blocking agent to the target-binding agentcan range from about 2:1 to about 100:1, from about 2:1 to about 50:1,or from about 5:1 to about 25:1. In some embodiments, the concentrationratio of the blocking agent to the target-binding agent can be at leastabout 100:1, at least about 1000:1, at least about 2500:1, at leastabout 5000:1, at least about 7500:1, or at least about 10,000:1.

In some embodiments, the blocking agent can be provided in thepre-determined concentration, which is sufficient to reduce the bindingof said at least one interfering agent to the target-binding agent,e.g., by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60% or more, ascompared to the binding in the absence of the blocking agent. Withoutwishing to be bound by theory, by reducing the binding of said at leastone interfering agent to the target-binding agent, the bindingsensitivity of the target binding agent for the corresponding targetentity can be increased, e.g., due to a lower background noisescontributed by the interfering agent. Accordingly, in some embodiments,the blocking agent can be provided in the pre-determined concentration,which is sufficient to decrease the lower limit of detection of thetarget-binding agent binding to the first target entity in the sample,e.g., by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60% or more, whencompared to the lower limit of detection in the absence of the blockingagent.

In some embodiments, the concentration of the blocking agent in a samplecan be selected to minimize non-specific binding (including, e.g.,binding of interfering agent(s) to a target-binding agent) whileminimizing inhibition of a target entity binding to a target-bindingagent.

In some embodiments, the blocking agent can be set to a concentrationthat is determined based on, e.g., the matrix composition of a sample(e.g., a blood sample) and/or concentrations of interfering agent(s) inthe sample. In some embodiments, the concentration of the blocking agentadded can increase with the amount/concentration of interfering agentspresent in a sample. In some embodiments, the concentration of theblocking agent added can increase with the amount/concentration ofinterfering agent(s) present in a sample, while the amount/concentrationof the target-binding agent remains about the same.

The pre-determined concentration of the blocking agent can vary with theeffective binding affinity of the target entity for the target-bindingagent. For example, a higher concentration of the blocking agent can beused without adversely affect the binding of the target-binding agent tothe target entity with a higher effective binding affinity. In someembodiments, the pre-determined concentration of the blocking agent canrange from about 1 mM to about 500 mM, or from about 5 mM to about 250mM, or from about 10 mM to about 100 mM.

In some embodiments where the glucose is the blocking agent, thepre-determined concentration of glucose can range from about 5 mM toabout 200 mM. In some embodiments, the pre-determined concentration ofglucose can be less than 20 mM. In some embodiments, the pre-determinedconcentration of glucose can be more than 20 mM.

In some embodiments, the method can further comprise separating thetarget-binding agent from the sample after the contacting. For example,the target-binding agent in the form of magnetic particles (e.g.,target-binding magnetic particles) can be separated from the sampleafter the contacting in the presence of a magnetic field gradient.

In some embodiments, the method can further comprise performing acompetitive wash to release, if any, said at least one interfering agentthat remains bound to the target-binding agent after the contactingand/or separation. For example, the target-binding agent after thecontacting and/or separation can be further washed with a buffercomprising a blocking agent described herein so as to remove anyresidual interfering agents still bound to the target-binding agent. Theblocking agent used in the wash buffer can be the same as, or differentfrom, the one used during the capture of the target entity.

In some embodiments, the method can further comprise detecting thepresence or absence of a target entity, after the sample is contactedwith a composition comprising a target-binding agent and a blockingagent. The target entity, if present, can remain bound on thetarget-binding agent during the detection, or be detached from thetarget-binding agent prior to the detection. Methods for detecting thetarget entity are known in the art. For example, in some embodiments,the target entity can be detected by a method comprising contacting thebound or detached target entity with a detection agent.

In some embodiments, the method can further comprise detecting thedisplaced blocking agent. Without wishing to be bound by theory, theamount of the displaced blocking agent can be proportional to the amountof the target entity displacing the blocking agent. Accordingly, in someembodiments, rather than directly determining the amount of the targetentity bound on the target-binding agent, the amount of the targetentity can be also reflected by a measurement of the amount of thedisplaced blocking agent. To facilitate detection of the displacedblocking agent, in some embodiments, the blocking agent can comprise adetectable label, e.g., a fluorescent label.

Various embodiments of the methods described herein can be adapted tovarious applications or be integrated as part of a process, e.g., butnot limited to, antibody-based assays (e.g., ELISA), filtrations,microbe detection and/or capture, antibiotic susceptibility testings,multiplexing assays, coating processes, hybridization-based assays,diagnostic strips, targeted drug delivery, or any combinations thereof.Thus, various compositions comprising a target-binding agent and atleast one blocking agent can be formulated to suit the need of eachindividual application. Accordingly, another aspect provided hereinrelates to a composition comprising one or more embodiments of atarget-binding agent described herein, and at least one embodiment of ablocking agent described herein at a pre-determined concentration,wherein the effective binding affinity of said at least one blockingagent for the target-binding agent is lower than the effective bindingaffinity of a target entity to be captured, and wherein the effectivebinding affinity of said at least one blocking agent for thetarget-binding agent is higher than the effective binding affinity of atleast one interfering molecule present in a sample to be assayed for thetarget-binding agent.

In some embodiments, said at least one blocking agent can be pre-boundto the target-binding agent within the composition. In alternativeembodiments, said at least one blocking agent and the target-bindingagent can be kept separately within the composition, e.g., each iscontained in a separate container. In some embodiments, thetarget-binding agent and said at least one blocking agent can each beindependently present in a buffered solution.

In some embodiments, the composition can further comprise a solidsubstrate affixed with at least one target-binding agent. Examples ofthe solid substrate include, but are not limited to, a nucleic acidscaffold, a protein scaffold, a lipid scaffold, a dendrimer,microparticle or a microbead, a nanotube, a microtiter plate, a medicalapparatus or implant, a microchip, a filtration device, a membrane, adiagnostic strip, a dipstick, an extracorporeal device, a mixing element(e.g., a spiral mixer), a microscopic slide, a hollow fiber, a hollowfiber cartridge, and any combinations thereof. In some embodiments, thecomposition can comprise microparticle or a microbead (e.g., polymericparticle and/or magnetic particle) affixed with the target-bindingagent. In some embodiments, the composition can comprise a dipstickaffixed with the target-binding agent.

In some embodiments, the target-binding agent can comprise an antibody(e.g., a primary antibody, and/or a secondary antibody). In theseembodiments, by way of example only, the composition can be used duringimmunoglobulin secondary detection reactions, immunostaining, and/orELISA assay.

In some embodiments, the target-binding agent can comprise amicrobe-binding agent (e.g., FcMBL molecule). Examples ofmicrobe-binding agent for detection and/or capture of microbes and/orfragments thereof are known in the art, including, e.g., microbe-bindingmolecules disclosed herein and in the International Application Nos.WO/2011/090954 (corresponding U.S. patent application Ser. No.13/574,191 entitled “Engineered opsonin for pathogen detection andtreatment”) and WO/2013/012924 (corresponding U.S. patent applicationSer. No. 14/233,553 entitled “Engineered microbe-targeting molecules anduses thereof”), the content of which are incorporated herein byreference.

In some embodiments where the microbe-binding agent comprises amannan-binding domain (e.g., FcMBL molecule), said at least one blockingagent can comprise glucose, maltose, N-acetyl muramic acid, and/or anycombinations thereof. The pre-determined concentration of the blockingagent can vary with the binding affinity of the target entity for thetarget-binding agent. For example, a higher concentration of theblocking agent can be used without adversely affect the binding of thetarget-binding agent to the target entity with a higher effectivebinding affinity. In some embodiments where the glucose is the blockingagent, the pre-determined concentration of glucose can range from about5 mM to about 200 mM.

In some embodiments, the blocking agent can further comprise adetectable label.

A kit comprising at least one composition described herein is alsoprovided. In some embodiments, the kit comprises a first compositioncomprising a first target-binding agent and at least one first blockingagent at a first pre-determined concentration, wherein the effectivebinding affinity of said at least one first blocking agent for the firsttarget-binding agent is lower than the effective binding affinity of afirst target entity to be captured, and wherein the effective bindingaffinity of said at least one first blocking agent for the firsttarget-binding agent is higher than the effective binding affinity of atleast one first interfering molecule present in a sample to be assayedfor the first target-binding agent; and instructions for using thecomposition for detecting or capturing the first target entity.

In some embodiments, the kit can further comprise a second compositioncomprising a second target-binding agent and at least one secondblocking agent at a second pre-determined concentration, wherein theeffective binding affinity of said at least one second blocking agentfor the second target-binding agent is lower than the effective bindingaffinity of a second target entity to be captured, and wherein theeffective binding affinity of said at least one second blocking agentfor the second target-binding agent is higher than the effective bindingaffinity of at least one second interfering molecule present in thesample to be assayed for the second target-binding agent.

In some embodiments, the first blocking agent and the second blockingagent can be selected to prevent or reduce the binding of the sameinterfering agent to the first target-binding agent and the secondtarget-binding agent, respectively. By way of example only, where saidat least the first interfering agent and/or said at least the secondinterfering agent can be a non-specific binding molecule present in asample, the first blocking agent and the second blocking agent can beselected to prevent or reduce the binding of non-specific bindingmolecules to the first target-binding agent and the secondtarget-binding agent, respectively.

In some embodiments, the first blocking agent and the second blockingagent can be selected to prevent or reduce the binding of a differentinterfering agent to the first target-binding agent and the secondtarget-binding agent, respectively. By way of example only, where thekit is adapted for use in a multiplexing assay, a first target entitycan be intended to be detected by a first target-binding agent but not asecond target-binding agent, while a second target entity can beintended to be detected by a second target-binding agent, but not afirst target-binding agent. In these embodiments, the first targetentity can be considered as said second interfering agent with respectto binding interaction between the second target-binding agent and thesecond target entity, and the second target entity can be considered assaid first interfering agent with respect to binding interaction betweenthe first target-binding agent and the first target entity.

In some embodiments, the first blocking agent can be pre-bound to thefirst target-binding agent. In some embodiments, the second blockingagent can be pre-bound to the second target-binding agent.

In some embodiments, the first target-binding agent can be affixed to afirst solid substrate. In some embodiments, the first solid substratecan be further affixed with the second target-binding agent. In someembodiments, the second target-binding agent can be affixed to a secondsolid substrate. Non-limiting examples of the first or the second solidsubstrate includes, but are not limited to, a nucleic acid scaffold, aprotein scaffold, a lipid scaffold, a dendrimer, microparticle or amicrobead, a nanotube, a microtiter plate, a medical apparatus orimplant, a microchip, a filtration device, a membrane, a diagnosticstrip, a dipstick, an extracorporeal device, a mixing element (e.g., aspiral mixer), a microscopic slide, a hollow fiber, a hollow fibercartridge, and any combination thereof.

In some embodiments, the kit can further comprise a first detectionagent capable of binding to the first target entity. In someembodiments, the kit can further comprise a second detection agentcapable of binding to the second target entity.

The methods, compositions and kits described herein can be applicablefor use with any sample. For example, a sample can comprise, withoutlimitations, a biological sample (e.g., bodily fluids such as blood,cells, and tissue samples), an environmental sample, a cell culturesample, a blood culture, water, pharmaceutical preparations, foods,beverages, and any combinations thereof. In some embodiments, the sampleis a fluid sample, e.g., blood or serum.

In some embodiments, the sample can comprise or be attached to a solidsubstrate as described herein. For example, in one embodiment, a samplecan comprise a biological sample (e.g., a tissue sample) on amicroscopic slide. In another embodiment, a sample can comprise proteinor peptide on a membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing effects of adding various concentrationsof a blocking agent (e.g., glucose) on binding of a target entity (e.g.,mannan) to a target-binding agent (e.g., FcMBL beads) in a buffer. FIG.1 shows that the addition of ˜10 mM glucose does not adversely affectmannan binding in buffer, whereas ˜20 mM glucose effectively reduces themannan binding by ˜50% and ˜40 mM glucose almost abolishes the mannanbinding in buffer.

FIG. 2 is a line graph showing effects of adding various concentrationsof a blocking agent (e.g., glucose) on binding of a target entity (e.g.,lipopolysaccharide (LPS)) to a target-binding agent (e.g., FcMBL beads)in a buffer. FIG. 2 shows that higher concentrations of glucose arerequired to effectively compete for binding of LPS to FcMBL. Unlikemannan detection (shown in FIG. 1), the addition of ˜40 mM glucose or˜80 mM glucose does not significantly affect the detection of LPS inserum. When glucose was added at a concentration of about 160 mM, theLPS detection was reduced to 10% as compared to the LPS level determinedin the absence of glucose.

FIGS. 3A-3B are data graphs showing effects of adding a blocking agent(e.g., ˜10 mM glucose) on binding of a target entity (e.g., LPS) or aninterfering agent (e.g., haemocytes) to a target-binding agent (e.g.,FcMBL beads) in donor blood. FIG. 3A shows that the addition of glucosereduces the background noise contributed by interfering agents presentin donor blood, thereby increasing the specificity of FcMBL binding toLPS, as evidenced by decreasing OD₄₅₀ signal as the concentration of LPSspiked in donor blood decreases. Further, FIG. 3B shows that theaddition of glucose significantly decreases the binding of haemocytes(e.g., erythrocytes) to FcMBL in donor blood.

FIG. 4 is data graph showing effects of adding a blocking agent (e.g.,˜10 mM glucose) on binding of a microbe or a fragment thereof to atarget-binding agent (e.g., FcMBL) in clinical samples. The microbialdetection was determined by FcMBL ELISA.

FIG. 5 is a schematic of an exemplary process comprising capture anddetection of a microbe and/or microbial fragments/matter in a testsample.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of various aspects described herein relate to methods,compositions and kits for detecting or capturing at least one targetentity. The inventors have discovered inter alia that a target-bindingagent pre-bound with an appropriate concentration of anintermediate-affinity ligand, prior to contacting a sample with thetarget-binding agent, can reduce non-target binding during capture of atarget entity in the sample (e.g., blood). A traditional blocking agent(e.g., bovine serum albumin, which has no binding specificity) has beencommonly used to mask unoccupied binding sites on an interfering agent(non-target material). However, the blocking agent described herein isunique, as the blocking agent is selected to bind specifically to atarget-binding agent, not any other material such as interfering agents,and also be capable of being displaced by a target entity, if present ina sample. In particular, the inventors have discovered that, in oneembodiment, pre-treating a microbe-binding agent comprising amannan-binding domain (e.g., FcMBL) with an intermediate-affinityblocking agent (e.g., glucose) can not only improve binding specificityand/or sensitivity of the microbe-binding agent for microbes and/orfragments thereof (target entity), but can also reduce false positivesresulting from non-target binding (e.g., haemocyte binding). In theseembodiments, glucose was selected as one of the blocking agents for usein the FcMBL system to detect and/or capture microbes or fragmentsthereof, partly because glucose can prevent non-target or interferingagents such as haemocytes from binding to FcMBL, while permitting atarget entity such as microbes and/or fragments thereof to displaceglucose that is bound to FcMBL.

The concept of pre-treating a target-binding agent with a blockingagent, where the blocking agent is selected based on relative bindingaffinities of the blocking agent, a target entity and an interferingagent, respectively, for the target-binding agent, can be extended toany detection/capture processes, assays, systems and/or platforms inwhich binding interaction between the target entity and thetarget-binding agent is involved in a matrix comprising at least oneinterfering agent. The blocking agent used in these detection/captureprocesses, assays, systems and/or platforms can be selected to have aneffective binding affinity for the target binding agent that is betweenan effective binding affinity of a target entity for the target-bindingagent and the effective binding affinity of an interfering agent for thetarget-binding agent, so that the target entity, but not the interferingagent, can displace the blocking agent that is bound to thetarget-binding agent, and thus be captured on the target-binding agent.

Methods of Detecting or Capturing at least one Target Entity

In one aspect, provided herein relates to methods of detecting orcapturing at least one target entity, including, e.g., at least twotarget entities, at least three target entities, or more. The methodcomprises contacting a sample with a composition comprising atarget-binding agent and a blocking agent, wherein the blocking agent isselected for reducing the binding of at least one interfering agentpresent in the sample to the target-binding agent, while permitting afirst target entity, if present in the sample, to (a) displace theblocking agent bound to the target-binding agent, or to (b) bind to thetarget-binding agent without the blocking agent bound thereto.

In some embodiments, the blocking agent is bound to the target-bindingagent, prior to contacting the sample with the composition. As usedherein, the term “binding” or “bound” generally refers to a reversiblebinding of one agent or molecule to another agent or molecule via, e.g.,van der Waals force, hydrophobic force, hydrogen bonding, and/orelectrostatic force. The binding interaction between an agent ormolecule and another agent or molecule can be described by adissociation constant (K_(d)) or association constant (K), which isfurther described below. For example, in the presence of a higheraffinity binder (e.g., a target entity), the blocking agent can bedisplaced by the higher affinity binder (e.g., a target entity).

In some embodiments, the blocking agent and the target-binding agent canbe concurrently added to a sample.

The term “displace” is used in reference to a target entity beingcapable of causing a blocking agent that is bound to the target-bindingagent to be released from the target-binding agent in order for thetarget entity to bind with the target-binding agent. The displacement ofthe blocking agent by the target-binding agent will generally occur whenthe target entity has a higher effective binding affinity for thetarget-binding agent than the blocking agent for the target-bindingagent, e.g., by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or more. Insome embodiments, the displacement of the blocking agent by thetarget-binding agent will occur when the target entity has a highereffective binding affinity for the target-binding agent than theblocking agent for the target-binding agent, e.g., by at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, or more.

The term “contacting” or “contact” as used herein in connection withcontacting a sample refers to subjecting a sample to a compositioncomprising a target-binding agent and a blocking agent by any means,which can vary with different formats of the compositions. For example,in some embodiments, the composition can be added into a fluid sample.In some embodiments, the sample can be flown through the composition(e.g., as a membrane) described herein to make the contact. In someembodiments, the sample can flow through a channel, e.g., a microfluidicchannel or a tubing, having the inner wall coated with the compositiondescribed herein to make the contact. In some embodiments, the samplecan be deposited or placed on the composition (e.g., a compositioncomprising a solid substrate affixed with the target-binding agent and ablock agent bound thereto).

In some embodiments of this aspect and other aspects described herein,the effective binding affinity of the blocking agent for thetarget-binding agent can be selected for increasing apparent specificityof the target-binding agent to the first target entity in the sample, ascompared to the apparent specificity in the absence of the blockingagent. For example, the apparent specificity of the target-binding agentto the first target entity can be increased by reducing the binding ofsaid at least one interfering agent to the target-binding agent, whichcan in turn increase the availability of binding sites on thetarget-binding agent for the target entity. While the binding of said atleast one interfering agent to the target-binding agent is typicallyreduced by blocking the unoccupied binding sites on the interferingagent, the inventors instead pre-block the binding sites on thetarget-binding agent with a blocking agent such that the interferingagent cannot bind to the target-binding agent, but the target entity isable to displace the blocking agent due to the target entity's higheraffinity for the target-binding agent than that of the blocking agent.Selection of an appropriate blocking agent for use in the methodsdescribed herein is described in the section “Blocking agents” laterbelow.

In some embodiments, the method can further comprise exposing thetarget-binding agent to the blocking agent at a pre-determinedconcentration to form the composition comprising the target-bindingagent and the blocking agent bound thereto, prior to the contacting ofthe sample with the composition.

The blocking agent can be generally present in a sample at anyconcentration provided that its presence in the sample does notadversely affect the binding of the first target entity to thetarget-binding agent. In some embodiments, the blocking agent can bepresent in a pre-determined concentration that does not reduce thebinding of the first target entity to the target-binding agent, e.g., bymore than 60%, more than 50%, more than 40%, more than 30%, more than20%, more than 10%, more than 5%, more than 1%, or less, as compared tothe binding in the absence of the blocking agent.

FIG. 3A shows that the addition of a blocking agent (e.g., glucose)reduces the background noise contributed by interfering agents presentin donor blood, thereby increasing the specificity of a target-bindingagent (e.g., FcMBL) binding to a target entity (e.g., LPS), as evidencedby decreasing OD₄₅₀ signal as the concentration of LPS spiked in donorblood decreases. Further, FIG. 3B shows that the addition of a blockingagent (e.g., glucose) can significantly decrease the binding ofinterfering agents such as haemocytes (e.g., erythrocytes) to atarget-binding agent (e.g., FcMBL) in donor blood. Accordingly, in someembodiments, the blocking agent can be provided in the pre-determinedconcentration, which is sufficient to reduce the binding of said atleast one interfering agent to the target-binding agent, e.g., by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or more, as compared to the binding in theabsence of the blocking agent. In one embodiment, the blocking agent canbe present in a concentration sufficient to partially or completelyinhibit the binding of said at least one interfering agent to thetarget-binding agent. Without wishing to be bound by theory, by reducingor inhibiting the binding of said at least one interfering agent to thetarget-binding agent, the binding sensitivity and/or specificity of thetarget binding agent for the corresponding target entity can beincreased, e.g., due to a lower background noise contributed by theinterfering agent. Accordingly, in some embodiments, the blocking agentcan be provided in the pre-determined concentration, which is sufficientto decrease the lower limit of detection of the target-binding agentbinding to the first target entity in the sample, e.g., by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60% , at least about 70%, at leastabout 80%, at least about 90%, or more, when compared to the lower limitof detection in the absence of the blocking agent.

The pre-determined concentration of the blocking agent can vary with theeffective binding affinity of the target entity for the target-bindingagent. For example, a target-binding agent (e.g., FcMBL) has arelatively high affinity for bacterial lipopolysaccharide (LPS), ascompared to mannan binding. Thus, as shown in FIG. 2, higherconcentrations of a blocking agent (e.g., glucose) are required toeffectively compete with LPS for binding to FcMBL. Unlike mannandetection as shown in FIG. 1, the addition of ˜40 mM blocking agent(e.g., glucose) or ˜80 mM blocking agent (e.g., glucose) does notsignificantly affect the detection of a target entity (LPS) in serum,until high concentrations of the blocking agent were used. Accordingly,a higher concentration of the blocking agent can be used withoutadversely affect the binding of the target-binding agent to the targetentity with a higher effective binding affinity. In some embodimentswhere the glucose is the blocking agent, the pre-determinedconcentration of glucose can range from about 5 mM to about 200 mM. Insome embodiments, the pre-determined concentration of glucose can beless than 20 mM. In some embodiments, the pre-determined concentrationof glucose can be more than 20 mM. One of skill in the art can determinethe optimum concentration of the blocking agent to be used, e.g., bydetermining amounts of target entity bound to the target-binding agentin the presence of blocking agent at various concentrations, e.g., asdescribed in Example 1 and shown in FIGS. 1-2.

In some embodiments, the method can further comprise separating thetarget-binding agent from the sample after the contacting. By way ofexample only, the target-binding agent in the form of magnetic particles(e.g., target-binding magnetic particles) can be separated from thesample after the contacting in the presence of a magnetic fieldgradient. In some embodiments where the target-binding agent is atarget-binding dipstick, target-binding agent can be separated from thesample by simply removing the target-binding dipstick from the sample.In some embodiments where the target-binding agent comprises particles,the target-binding agent can be separated from the sample bycentrifugation and/or filtration. Methods for separating thetarget-binding agent from the sample are dependent on the format of thetarget-binding agents and are generally known in the art. In someembodiments where the method described herein is used to detect and/orcapture a microbe, the target-binding agent (e.g., a microbe-bindingagent) can be separated from the sample following one or a combinationof the methods as described in step 1210 (microbe separation) of theprocess 1200 described later below.

In some embodiments, the method can further comprise performing acompetitive wash to release, if any, said at least one interfering agentthat remains bound to the target-binding agent after the contactingand/or separation. For example, the target-binding agent after thecontacting and/or separation can be further washed with a buffercomprising a blocking agent described herein so as to remove anyresidual interfering agents still bound to the target-binding agent. Insome embodiments, the competitive wash can be used to remove anyresidual interfering agents not competed away by the target-bindingagent, e.g., due to low abundance of the target binding agent. Theblocking agent used in the wash buffer can be the same as, or differentfrom, the one used during the capture of the target entity.

In some embodiments, the method can further comprise detecting a targetentity, after the sample is contacted with a composition comprising atarget-binding agent and a blocking agent. The target entity can remainbound on the target-binding agent during detection and/or analysis, orbe detached from the target-binding agent prior to detection and/oranalysis. Methods for detecting the target entity are known in the art,e.g., by spectroscopy, electrochemical detection, polynucleotidedetection, fluorescence anisotropy, fluorescence resonance energytransfer, electron transfer, enzyme assay, magnetism, electricalconductivity, isoelectric focusing, chromatography, immunoprecipitation,immunoseparation, aptamer binding, filtration, electrophoresis, use of aCCD camera, immunoassay, ELISA, Gram staining, immunostaining,microscopy, immunofluorescence, western blot, polymerase chain reaction(PCR), RT-PCR, fluorescence in situ hybridization, sequencing, massspectroscopy, or substantially any combination thereof.

In some embodiments, the detecting of a target entity can comprisecontacting the target-binding agent with a detection agent, wherein thetarget entity, if present, remains bound on the target-binding agent. Insome embodiments, a sub-population of the target-binding agent comprisesa target entity, if present in a sample, bound thereto; while asub-population of the target-binding agent can still comprise theblocking agent bound thereto. In some embodiments, a sub-population ofthe target-binding agent can comprise both the target entity and theblocking agent bound thereto. In some embodiments, the blocking agentthat is already bound to the target-binding agent cannot bind to adetection agent and thus cannot interfere with detection of the boundtarget entity. In other embodiments, the blocking agent that is alreadybound to the target-binding agent can still bind to a detection agent.In these embodiments, the blocking agent can be treated, prior toaddition of a detection gent, to mask its spare binding sites such thatthe blocking agent becomes unable to bind to the detection agent.

In some embodiments, the detecting of a target entity can comprisecontacting a target entity, if present, with a detection agent, whereinthe target entity has been detached from the target-binding agent.

The detection agent used to detect a target entity can be any moleculeor compound that can bind to a target entity and be detected by anymethods known in the art. Examples of a detection agent include, but arenot limited to, proteins, peptides, antibodies or fragments thereof,aptamers, nucleic acid molecules, polynucleotides, oligonucleotides,carbohydrates, and any combinations thereof. In some embodiments, thedetection agent can comprise a detectable label. A detectable label caninclude, but not limited to, an optical label, a radioactive label, anoligonucleotide label, an enzyme label (e.g., but not limited to, ahorseradish peroxidase (HRP) or an alkaline phosphatase (AP)), ametabolic label, or any combinations thereof.

In some embodiments where the target entity comprises a microbe or afragment thereof, the detection agent can comprise a carbohydraterecognition domain derived from a carbohydrate-binding molecule.Examples of a carbohydrate-binding molecule include, but are not limitedto, lectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, glucose-bindingprotein, Galanthus nivalis agglutinin, peanut lectin, lentil lectin,DC-SIGN, C-reactive protein, and any combinations thereof. In someembodiments, the detection agent can comprise a carbohydrate recognitiondomain and a detectable label. In some embodiments, the detection agentis a fusion peptide comprising a carbohydrate recognition domain of alectin, wherein the fusion peptide is conjugated to a detectable label.For example, the fusion peptide can be a FcMBL, which is a fusionpeptide comprising mannan-binding lectin and a Fc portion of animmunoglobulin, and is described in the U.S. application Ser. No.13/574,191 entitled “Engineered Opsonin for Pathogen Detection andTreatment” and U.S. application Ser. No. 14/233,553 entitled “EngineeredMicrobe-Targeting Molecules and Uses Thereof,” both of which the patentapplications are incorporated herein by reference. In some embodiments,a detection agent can be a FcMBL conjugated to an enzyme label (e.g.,but not limited to, a horseradish peroxidase or an alkalinephosphatase). Detection agents such as FcMBL-HRP or FcMBL-AP describedin U.S. application Ser. No. 14/233,553 entitled “EngineeredMicrobe-Targeting Molecules and Uses Thereof,” incorporated byreference, can be also used herein.

In some embodiments, the method can further comprise detecting thedisplaced blocking agent. Without wishing to be bound by theory, theamount of the displaced blocking agent can be proportional to the amountof the target entity displacing the blocking agent. Accordingly, in someembodiments, rather than directly determining the amount of the targetentity bound on the target-binding agent, the amount of the targetentity can be also reflected by a measurement of the amount of thedisplaced blocking agent. To facilitate detection of the displacedblocking agent, in some embodiments, the blocking agent can comprise adetectable label, e.g., a fluorescent label or any detectable labelsdescribed herein.

Blocking Agents

While it may not be necessary to have a target-binding agent pre-boundwith a blocking agent, in some embodiments, it can be beneficial to addthe blocking agent to the target-binding agent prior to contacting asample with the target-binding agent, e.g., when there is a much higherabundance of interfering agents than the target entity present in asample.

In some embodiments, the blocking agent can be selected based on itseffective binding affinity relative to effective binding affinities ofthe first target entity and said at least one interfering agent,respectively, for the target-binding agent. In some embodiments, theblocking agent can be selected to have an effective binding affinity forthe target-binding agent that is lower than an effective bindingaffinity of the first target entity for the target-binding agent; andthe blocking agent is further selected to have the effective bindingaffinity for the target-binding agent that is higher than an effectivebinding affinity of at least one interfering agent present in the samplefor the target-binding agent.

In order to permit the first target entity to displace the blockingagent bound to the target-binding agent, the effective binding affinityof the blocking agent for the target-binding agent is desired to belower than the effective binding affinity of the first target entity forthe target-binding agent. In some embodiments of this aspect and otheraspects described herein, the effective binding affinity of the blockingagent for the target-binding agent can be lower than the effectivebinding affinity of the first target entity for the target-binding agentby at least about 10% or more, including, e.g., at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or more.

In order to reduce or prevent at least one interfering agent frombinding to the target-binding agent, the effective binding affinity ofthe blocking agent for the target-binding agent is desired to be higherthan the effective binding affinity of said at least one interferingagent present in the sample for the target-binding agent. In someembodiments of this aspect and other aspects described herein, theeffective binding affinity of the blocking agent for the target-bindingagent can be higher than the effective binding affinity of said at leastone interfering agent present in the sample for the target-binding agentby at least about 10% or more, including, e.g., at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90% or more.In some embodiments, the effective binding affinity of the blockingagent for the target-binding agent can be higher than the effectivebinding affinity of said at least one interfering agent present in thesample for the target-binding agent by at least about 1-fold or more,including, e.g., at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold or more.

As used herein, the term “effective binding affinity” generally refersto an overall binding property of a first agent (e.g., a blocking agent,an interfering agent, and/or a target entity) interacting with a secondagent (e.g., a target-binding agent) under a specific condition, and theoverall binding property is typically dependent on intrinsiccharacteristics of the first agent and the second agent including, butnot limited to, surface composition of the first agent and/or the secondagent (e.g., but not limited to, density of target-binding moleculespresent on the surface of the target-binding agent such as FcMBL densityon the beads), single-bond affinity, avidity, as well as thesurrounding/ambient condition for the binding interaction, e.g., but notlimited to, concentration of the first agent and/or the second agent,and/or the presence of a third agent (e.g., a blocking agent, aninterfering agent and/or a target entity) during the binding interactionbetween the first and the second agents. Different measures of aneffective binding affinity of an agent are known in the art. In someembodiments, the effective binding affinity of a first agent for asecond agent can be indicated by a dissociation constant (K_(d)) forbinding of the first agent to the second agent. The dissociationconstant (K_(d)) is an equilibrium constant that generally measures thepropensity of a bound complex to separate (dissociate) reversibly intoseparate agents. In these embodiments, a higher dissociation constantindicates a lower effective binding affinity. Alternatively, theeffective binding affinity of a first agent for a second agent can beindicated by an association constant (K) for binding of the first agentto the second agent. The association constant (K) is the inverse of thedissociation constant (K_(d)), i.e., a higher association constantindicates a higher effective binding affinity.

As used herein, the term “single-bond affinity” refers to the strengthof a single bond interaction, including but not limited to hydrogenbonds, electrostatic bonds, van der Waals forces, hydrophobic forces, orany combinations thereof.

As used herein, the term “avidity” refers to the combined strength ofmultiple bond interactions. Avidity is distinct from affinity orsingle-bond affinity, which is the strength of a single bondinteraction. In general, avidity is the combined synergistic strength ofbond affinities rather than the sum of bonds. Accordingly, avidity isgenerally used to describe one agent having multiple interactions withanother agent. For example, where the blocking agent may have similarsingle-bond affinity as that of the target entity to the target-bindingagent, the target entity can have higher avidity than that of theblocking agent if the target entity can form more bonds than theblocking agent with the target-binding agent. The higher avidity of thetarget entity would yield a higher effective binding affinity than thatof the blocking agent.

Accordingly, in some embodiments, the effective binding affinity of theblocking agent can be dependent on a number of properties including, butnot limited to, surface composition of the blocking agent, avidity,single-bond affinity or affinity, surface composition of thetarget-binding agent (e.g., but not limited to, density oftarget-binding molecules present on the surface of the target-bindingagent), concentration of the blocking agent, concentration of the firsttarget entity in the sample, concentration of said at least oneinterfering agent, or any combinations thereof. By way of example only,in some embodiments where both the target entity and the blocking agentcomprise a glucose molecule, the target entity, e.g., a microbe or afragment thereof comprising on its surface a glucose molecule incombination with other sugar molecules, or more than one glucosemolecules in a specific pattern, can have a higher effective bindingaffinity for the target-binding agent (e.g., FcMBL) that that of aglucose monomer as a blocking agent, partly due to the higher avidityobserved in the microbes or fragments thereof.

In some embodiments of this aspect and other aspects described herein,the effective binding affinity of the blocking agent for thetarget-binding agent, as indicated by a dissociation constant for thebinding of the blocking agent to the target-binding agent, can rangefrom about 1 μM to about 100 mM, or about 10 μM to about 75 mM, or about5 mM to about 50 mM. In one embodiment, the effective binding affinityof the blocking agent for the target-binding agent, as indicated by adissociation constant for the binding of the blocking agent to thetarget-binding agent, can range from about 5 mM to about 50 mM. In someembodiments, the effective binding affinity of the blocking agent forthe target-binding agent, as indicated by a dissociation constant forthe binding of the blocking agent to the target-binding agent, can be ina nanomolar range. For example, in some embodiments, the effectivebinding affinity of the blocking agent for the target-binding agent, asindicated by a dissociation constant for the binding of the blockingagent to the target-binding agent, can range from about 1 nM to 10 μM,about 1 nM to about 1 μM, or about 10 nM to about 500 nM.

In some embodiments, the effective binding affinity ratio of a blockingagent to a target entity (for a target-binding agent), as indicated bydissociation constants, can range from about 2:1 to about 1000:1, fromabout 2:1 to about 500:1, from about 2:1 to about 100:1, or from about2:1 to about 50:1, from about 2:1 to about 25:1, from about 2:1 to about10:1, from about 2:1 to about 5:1.

The blocking agent can be any molecule, compound, or agent that can bindto a target-binding agent with an effective binding affinity betweenthat of the target entity and the interfering agent, respectively, forthe target-binding agent. Depending on the binding properties of thetarget-binding agents, target entity, and/or interfering agents, theblocking agent can include, but not limited to, cells or fragmentsthereof, peptides, polypeptides, proteins, peptidomimetics, antibodies,antibody fragments (e.g., antigen binding fragments of antibodies),carbohydrate-binding protein, e.g., lectins, glycoproteins,glycoprotein-binding molecules, amino acids, carbohydrates (includingmono-, di-, tri- and poly-saccharides), lipids, steroids, hormones,lipid-binding molecules, cofactors, nucleosides, nucleotides, nucleicacids (e.g., DNA or RNA, analogues and derivatives of nucleic acids, oraptamers), peptide aptamers, peptidoglycan, lipopolysaccharide, smallmolecules, endotoxins (e.g., bacterial lipopolysaccharide), and anycombinations thereof. In some embodiments, the cells include, but arenot limited to, prokaryotes (e.g., microbes such as bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, and fungi), bloodcells, and any fragments thereof. In some embodiments where thetarget-binding agent is configured to recognize a carbohydrate pattern,e.g., for detection and/or capture of a microbe or a fragment thereof,the blocking agent can be a carbohydrate or a saccharide.

In some embodiments, the blocking agent can be a monomer. In general, amonomer (with a single binding site) has no free binding site afterbinding to the target-binding agent. Accordingly, for monomeric blockingagents that have not been displaced by the target entity and stillremain bound on the target-binding agent, the bound monomeric blockingagents will unlikely be detected by an assay using a “sandwich” method,e.g., ELISA, due to the absence of binding sites for binding with adetection agent. Thus, the target entity can be detected, e.g., using a“sandwich” method, e.g., ELISA, without getting significant backgroundsignals from the monomeric blocking agents.

Examples of a saccharide-based monomeric blocking agent can be amonosaccharide or modification thereof, including, e.g., but not limitedto, diose, triose, tetrose, pentose, hexose, heptose, linear chainmonosaccharides, open chain monosaccharides, cyclic isomers (e.g.,furanose form and pyranose of monosaccharides such as hexose), pyranose,fructose, galactose, xylose, ribose, amino sugars (e.g., but not limitedto, galactosamine, glucosamine, sialic acid, N-acetylglucosamine,N-acetyl-muramic acid, sulfosugars (e.g., but not limited tosulfoquinovose). In some embodiments, one or more of thesesaccharide-based monomeric blocking agents can be used in an assay todetect and/or capture microbes and/or fragments thereof, e.g., addedprior to step 1208 (microbe capture) of process 1200 as described below.In one embodiment, the saccharide-based monomeric blocking agent cancomprise glucose.

In alternative embodiments, the blocking agent can be a multimer, i.e.,a blocking agent that has at least one free binding site after bindingto the target-binding agent. In these embodiments, bound multimerblocking agents remained on the target-binding agents can haveadditional binding sites. In some embodiments, addition of anothermonomeric blocking agent to block these additional binding sites priorto detection of the captured target entities, e.g., using a “sandwich”method such as ELISA or any other detection methods known in the art canbe performed. To identify K_(d) for multimers, one can, for example,evaluate the K_(d) of the corresponding monomers experimentally.

Examples of a saccharide-based multimeric blocking agent can be adisaccharide, an oligosaccharide, a polysaccharide, modificationsthereof, or any combinations thereof, including, e.g., but not limitedto, lactose, sucrose, maltose, lactulose, trehalose, cellobiose,kojibiose, nigerose, isomaltose, β, β-trehalose, α, β-trehalose,sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose,gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose,xylobiose, fructo-oligosaccharides, galactooligosaccharides, mannanoligosaccharides, cellulose, chitin, callose, laminarin,chrysilaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan,or any combinations thereof. In some embodiments, one or more of thesesaccharide-based multimeric blocking agents can be used in an assay todetect and/or capture microbes and/or fragments thereof, e.g., addedprior to step 1208 (microbe capture) of process 1200 as described below.

In some embodiments, a saccharide-based blocking agent can be glucose,maltose, N-acetyl-muramic acid, or any combinations thereof. In oneembodiment, a saccharide-based blocking agent can comprise glucose. Insome embodiments, one or more of these saccharide-based blocking agentscan be used in an assay to detect and/or capture microbes and/orfragments thereof, e.g., added prior to step 1208 (microbe capture) ofprocess 1200 as described below.

Interfering Agents

An interfering agent is an agent present in a sample to be assayed,which undesirably binds to a target-binding agent and reduces theeffective binding affinity of the target-binding agent to thecorresponding target entity, e.g., by at least about 10% or more,including, e.g., at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90% or more. Examples of the interferingagents include, but are not limited to, cells or fragments thereof,peptides, polypeptides, proteins, peptidomimetics, antibodies, antibodyfragments (e.g., antigen binding fragments of antibodies),carbohydrate-binding protein, e.g., lectins, glycoproteins,glycoprotein-binding molecules, amino acids, carbohydrates (includingmono-, di-, tri- and poly-saccharides), lipids, steroids, hormones,lipid-binding molecules, cofactors, nucleosides, nucleotides, nucleicacids (e.g., DNA or RNA, analogues and derivatives of nucleic acids, oraptamers), peptide aptamers, peptidoglycan, lipopolysaccharide, smallmolecules, endotoxins (e.g., bacterial lipopolysaccharide), and anycombinations thereof. In some embodiments, the cells include, but arenot limited to, prokaryotes (e.g., microbes such as bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, and fungi), bloodcells, and any fragments thereof.

In some embodiments where the sample comprises a biological fluid, e.g.,blood, at least one interfering agent can comprise a blood cell and/or afragment thereof, e.g., a red blood cell (or an erythryocyte) and/or afragment thereof.

In some embodiments where the sample comprises a second target entitynot intended to be captured or detected by the first target-bindingagent, but by a second target-binding agent, the second target entitycan be considered as an interfering agent with respect to the bindinginteraction between the first target-binding agent and the first targetentity.

In some embodiments, said at least one interfering agent can be anon-specific binding molecule. As used herein, the term “non-specificbinding molecule” refers to a molecule capable of binding to thetarget-binding agent, which is not correlated with the specificity ofthe target-binding agent. In some embodiments, the non-specific bindingmolecule can bind to the target-binding agent, e.g., via adsorption.

In some embodiments, the binding of said at least one interfering agentwith the target-binding agent can be specific, but the effective bindingaffinity of the interfering agent for the target-binding agent is lowerthan that of the target entity and/or the blocking agent for thetarget-binding agent.

In some embodiments, the effective binding affinity of said at least oneinterfering agent can be dependent on a number of properties including,but not limited to surface composition of the interfering agent,avidity, single-bond affinity or affinity, surface composition of thetarget-binding agent (e.g., but not limited to, density oftarget-binding molecules present on the surface of the target-bindingagent such as FcMBL density on the beads), concentration of the blockingagent, concentration of the first target entity in the sample,concentration of said at least one interfering agent, or anycombinations thereof.

As described herein, the effective binding affinity of said at least oneinterfering agent for the target-binding agent is lower than theeffective binding affinity of the blocking agent for the target-bindingagent. Accordingly, in some embodiments of this aspect and other aspectsdescribed herein, the effective binding affinity of said at least oneinterfering agent for the target-binding agent, as indicated by adissociation constant for the binding of the interfering agent to thetarget-binding agent, can be more than 5 μM, more than 10 μM, more than0.1 mM, more than 0.5 mM, more than 1 mM, more than 5 mM, more than 10mM, more than 25 mM, more than 50 mM, more than 75 mM, more than 100 mMor more, provided that the dissociation constant for the binding of theinterfering agent to the target-binding agent is larger than that forthe binding of the blocking agent to the target-binding agent. In someembodiments, the effective binding affinity of said at least oneinterfering agent for the target-binding agent, as indicated by adissociation constant for the binding of the interfering agent to thetarget-binding agent, can be more than 10 mM. In some embodiments, theeffective binding affinity of said at least one interfering agent forthe target-binding agent, as indicated by a dissociation constant forthe binding of the interfering agent to the target-binding agent, can bemore than 50 mM. In some embodiments, the effective binding affinity ofsaid at least one interfering agent for the target-binding agent, asindicated by a dissociation constant for the binding of said at leastone interfering agent to the target-binding agent, can be in a lowerrange, e.g., more than 500 nM, or more than 1 μM or higher.

Target Entity or Target Species

The methods, compositions and kits described herein can be used tocapture or isolate one or more target entities from a test sample. Asused interchangeably herein, the terms “target entity” and “targetspecies” refer to any molecule, cell or particulate that is to beseparated or isolated from a fluid sample. Representative examples oftarget cellular entities include, but are not limited to, mammaliancells, viruses, bacteria, fungi, yeast, protozoan, microbes, parasites,endotoxins (e.g., bacterial lipopolysaccharide), and any combinationsthereof. Representative additional examples of target entities include,but are not limited to, peptides, polypeptides, proteins,peptidomimetics, antibodies, antibody fragments (e.g., antigen bindingfragments of antibodies), carbohydrate-binding protein, e.g., lectins,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptide aptamers, peptidoglycan,lipopolysaccharide, small molecules, toxins, and any combinationsthereof. The target species can also include contaminants found innon-biological fluids, such as pathogens or lead in water or inpetroleum products. Parasites can include organisms within the phylaProtozoa, Platyhelminthes, Aschelminithes, Acanthocephala, andArthropoda.

In some embodiments, the effective binding affinity of the target entitycan be dependent on a number of properties including, but not limitedto, surface composition of the target entity, avidity, single-bondaffinity or affinity, surface composition of the target-binding agent(e.g., but not limited to, density of target-binding molecules presenton the surface of the target-binding agent such as FcMBL density on thebeads), concentration of the blocking agent, concentration of the firsttarget entity in the sample, concentration of said at least oneinterfering agent, or any combinations thereof.

As described earlier, the effective binding affinity of the blockingagent for the target-binding agent is lower than the effective bindingaffinity of the target entity for the target-binding agent. Accordingly,in some embodiments of this aspect and other aspects described herein,the effective binding affinity of the target entity for thetarget-binding agent, as indicated by a dissociation constant for thebinding of the target entity to the target-binding agent, can be lessthan 100 mM, less than 75 mM, less than 50 mM, less than 25 mM, lessthan 10 mM, less than 5 mM, less than 1 mM, less than 0.5 mM, less than0.1 mM, less than 10 μM, less than 1 μM, or less, provided that thedissociation constant for the binding of the blocking agent to thetarget-binding agent is smaller than higher than that for the binding ofthe target entity to the target-binding agent. In some embodiments, theeffective binding affinity of the target entity for the target-bindingagent, as indicated by a dissociation constant for the binding of thetarget entity to the target-binding agent, can be less than 25 mM. Insome embodiments, the effective binding affinity of the target entityfor the target-binding agent, as indicated by a dissociation constantfor the binding of the target entity to the target-binding agent, can bein a nanomolar range. For example, in some embodiments, the effectivebinding affinity of the target entity for the target-binding agent, asindicated by a dissociation constant for the binding of the targetentity to the target-binding agent, can be less than 1 μM, less than 500nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM,less than 5 nM, less than 1 nM, less than 0.5 nM, or less.

In some embodiments, the target species can include a biological cellselected from the group consisting of living or dead cells (prokaryoticand eukaryotic, including mammalian), viruses, bacteria, fungi, yeast,protozoan, microbes, and parasites. The biological cell can be a normalcell or a diseased cell, e.g., a cancer cell. Mammalian cells include,without limitation; primate, human and a cell from any animal ofinterest, including without limitation; mouse, hamster, rabbit, dog,cat, domestic animals, such as equine, bovine, murine, ovine, canine,and feline. In some embodiments, the cells can be derived from a humansubject. In other embodiments, the cells are derived from a domesticatedanimal, e.g., a dog or a cat. Exemplary mammalian cells include, but arenot limited to, stem cells, cancer cells, progenitor cells, immunecells, blood cells, fetal cells, and any combinations thereof. The cellscan be derived from a wide variety of tissue types without limitationsuch as, hematopoietic, neural, mesenchymal, cutaneous, mucosal,stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial,hepatic, kidney, gastrointestinal, pulmonary, cardiovascular, T-cells,and fetus. Stem cells, embryonic stem (ES) cells, ES-derived cells,induced pluripotent stem cells, and stem cell progenitors are alsoincluded, including without limitation, hematopoietic, neural, stromal,muscle, cardiovascular, hepatic, pulmonary, and gastrointestinal stemcells. Yeast cells may also be used as cells in some embodimentsdescribed herein. In some embodiments, the cells can be ex vivo orcultured cells, e.g. in vitro. For example, for ex vivo cells, cells canbe obtained from a subject, where the subject is healthy and/or affectedwith a disease. While cells can be obtained from a fluid sample, e.g., ablood sample, cells can also be obtained, as a non-limiting example, bybiopsy or other surgical means know to those skilled in the art.

In some embodiments, the target species refers to a rare cell or acellular component thereof. In some embodiments, the target species canrefer to a rare cell or a cellular component thereof derived from amammalian subject, including, without limitation, primate, human or anyanimal of interest such as mouse, hamster, rabbit, dog, cat, domesticanimals, such as equine, bovine, murine, ovine, canine, and feline. Insome embodiments, the rare cells can be derived from a human subject. Inother embodiments, the rare cells can be derived from a domesticatedanimal or a pet such as a cat or a dog. As used herein, the term “rarecells” is defined, in some embodiments, as cells that are not normallypresent in a fluid sample, e.g., a biological fluid sample, but can bepresent as an indicator of an abnormal condition, such as infectiousdisease, chronic disease, injury, proliferative diseases, or pregnancy.In some embodiments, the term “rare cells” as used herein refers tocells that can be normally present in biological specimens, but arepresent with a frequency several orders of magnitude (e.g., at leastabout 100-fold, at least about 1000-fold, at least about 10000-fold)less than other cells typically present in a normal biological specimen.In some embodiments, rare cells are found infrequently in circulatingblood, e.g., less than 100 cells (including less than 10 cells, lessthan 1 cell) per 10⁸ mononuclear cells in about 50 mL of peripheralblood. In some embodiments, a rare cell can be a normal cell or adiseased cell. Examples of rare cells include, but are not limited to,circulating tumor cells, progenitor cells, e.g., collected for bonemarrow transplantation, blood vessel-forming progenitor cells, stemcells, circulating fetal cells, e.g., in maternal peripheral blood forprenatal diagnosis, virally-infected cells, cell subsets collected andmanipulated for cell and gene therapy, and cell subpopulations purifiedfor subsequent gene expression or proteomic analysis, other cellsrelated to disease progression, and any combinations thereof.

As used herein, the term “a cellular component” in reference tocirculating tumor cells, stem cells, fetal cells and/or microbes isintended to include any component of a cell that can be at leastpartially isolated from the cell, e.g., upon lysis of the cell. Cellularcomponents can include, but are not limited to, organelles, such asnuclei, perinuclear compartments, nuclear membranes, mitochondria,chloroplasts, or cell membranes; polymers or molecular complexes, suchas lipids, polysaccharides, proteins (membrane, trans-membrane, orcytosolic); nucleic acids, viral particles, or ribosomes; or othermolecules, such as hormones, ions, and cofactors.

As used herein, the term “molecular toxin” refers to a compound producedby an organism which causes or initiates the development of a noxious,poisonous or deleterious effect in a host presented with the toxin. Suchdeleterious conditions may include fever, nausea, diarrhea, weight loss,neurologic disorders, renal disorders, hemorrhage, and the like. Toxinsinclude, but are not limited to, bacterial toxins, such as choleratoxin, heat-liable and heat-stable toxins of E. coli, toxins A and B ofClostridium difficile, aerolysins, and hemolysins; toxins produced byprotozoa, such as Giardia; toxins produced by fungi. Molecular toxinscan also include exotoxins, i.e., toxins secreted by an organism as anextracellular product, and enterotoxins, i.e., toxins present in the gutof an organism.

Target-Binding Agents

A target-binding agent is an agent configured to detect and/or captureat least one target entity described herein. The target-binding agentcan be present in any form, including but not limited to atarget-binding molecule, and/or a target-binding substrate (e.g., atarget-binding molecule conjugated to a solid substrate or a solidsupporting structure). In some embodiments, the target-binding agent cancomprise a target-binding molecule selected from the group consisting ofcells or fragments thereof, peptides, polypeptides, proteins,peptidomimetics, antibodies, antibody fragments (e.g., antigen bindingfragments of antibodies), carbohydrate-binding protein, e.g., lectins,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptide aptamers, peptidoglycan,lipopolysaccharide, small molecules, endotoxins (e.g., bacteriallipopolysaccharide), and any combinations thereof.

In some embodiments, a target-binding agent can comprise amicrobe-binding agent as described in the section “Microbe-bindingagents or molecules” below.

In some embodiments, the target-binding agent can be affixed to a solidsubstrate described herein to form a target-binding substrate.Non-limiting examples of a solid substrate include, but are not limitedto, a nucleic acid scaffold, a protein scaffold, a lipid scaffold, adendrimer, microparticle or a microbead, a nanotube, a microtiter plate,a medical apparatus or implant, a microchip, a filtration device, amembrane, a diagnostic strip, a dipstick, an extracorporeal device, amixing element (e.g., a spiral mixer), a microscopic slide, a hollowfiber, a hollow fiber cartridge, and any combinations thereof.

The solid substrate can be made of any material, including, but notlimited to, metal, metal alloy, polymer, plastic, paper, glass, fabric,packaging material, biological material such as cells, tissues,hydrogels, proteins, peptides, nucleic acids, and any combinationsthereof.

In some embodiments, the solid substrate can be fabricated from orcoated with a biocompatible material. As used herein, the term“biocompatible material” refers to any material that does notdeteriorate appreciably and does not induce a significant immuneresponse or deleterious tissue reaction, e.g., toxic reaction orsignificant irritation, over time when implanted into or placed adjacentto the biological tissue of a subject, or induce blood clotting orcoagulation when it comes in contact with blood. Suitable biocompatiblematerials include, for example, derivatives and copolymers ofpolyimides, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine,and polyvinylamine, polyacrylates, polyamides, polyesters,polycarbonates, and polystyrenes. In some embodiments, biocompatiblematerials can include metals, such as titanium and stainless steel, orany biocompatible metal used in medical implants. In some embodiments,biocompatible materials can include paper substrate, e.g., as a solidsubstrate for a diagnostic strip. In some embodiments, biocompatiblematerials can include peptides or nucleic acid molecules, e.g., anucleic acid scaffold such as a 2-D DNA sheet or 3-D DNA scaffold.

Additional material that can be used to fabricate or coat a solidsubstrate include, without limitations, polydimethylsiloxane, polyimide,polyethylene terephthalate, polymethylmethacrylate, polyurethane,polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon,polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene,polyacrylonitrile, polybutadiene, poly(butylene terephthalate),poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol),styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinylbutyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and anycombination thereof.

In some embodiments, at least a portion of a solid substrate surface tobe in contact with a test sample can be treated or modified to becomeless adhesive or non-adhesive to molecules present in the test sample.By way of example only, the solid substrate surface can be silanized orcoated with a polymer such that the solid substrate surface becomesinert and non-adhesive to any molecules present in a test sample. Inother embodiments, a solid substrate surface can be modified or overlaidwith a repellant or slippery surface. For example, a solid substratesurface can comprise a nano/microstructured substrate layer infused witha lubricating fluid, where the lubricating fluid is substantiallyimmobilized on the substrate layer to form a repellant or slipperysurface. In some embodiments, the repellant or slippery surface is knownas Slippery Liquid-Infused Porous Surface (SLIPS), which is described inWong T. S. et al., “Bioinspired self-repairing slippery surfaces withpressure-stable omniphobicity.” (2011) Nature 477 (7365): 443-447, andInternational Application Nos. PCT/US12/21928 and PCT/US12/21929, thecontents of which are incorporated herein by reference.

As used herein, by the “coating” or “coated” is generally meant a layerof molecules or material formed on a surface of a solid substrate. Withrespect to a coating of target-binding molecules on a solid substrate,the term “coating” or “coated” refers to a layer of target-bindingmolecules formed on a surface of a solid substrate. In some embodiments,the solid substrate surface can encompass an outer substrate surfaceand/or an inner substrate surface, e.g., with respect to a hollowstructure. For example, the inner surface of a needle or catheter can becoated with target-binding molecules described herein. In oneembodiment, the inner surface and/or outer surface of a hollow fiber canbe coated with target-binding molecules described herein. In someembodiments, a cartridge can comprise a plurality of (e.g., at least 2or more, including, e.g., at least 3, at least 4, at least 5, at least10, at least 20, or more) hollow fibers having their inner surfaceand/or outer surface coated with target-binding molecules describedherein. In one embodiment, the target-binding molecules coating a solidsubstrate surface can comprise microbe-binding molecules, e.g., forremoving any microbe contaminants or fragments thereof from a fluid.

A solid substrate surface can be functionalized or activated forconjugation with target-binding molecules by any methods known in theart. Exemplary conjugations include, but are not limited to, covalentbond, amide bond, additions to carbon-carbon multiple bonds, azidealkyne Huisgen cycloaddition, Diels-Alder reaction, disulfide linkage,ester bond, Michael additions, silane bond, urethane, nucleophilic ringopening reactions: epoxides, non-aldol carbonyl chemistry, cycloadditionreactions: 1,3-dipolar cycloaddition, temperature sensitive, radiation(IR, near-IR, UV) sensitive bond or conjugation agent, pH-sensitive bondor conjugation agent, non-covalent bonds (e.g., ionic charge complexformation, hydrogen bonding, pi-pi interactions, cyclodextrin/adamantlyhost guest interaction) and the like. In some embodiments, a solidsubstrate surface can be functionalized with addition of silane couplingagents (e.g., but not limited to organosilanes, aminosilanes, vinylsilanes, methacryl silanes, and any combinations thereof).

Target-binding magnetic particles: In some embodiments, thetarget-binding agents can comprise target-binding magnetic particles. Asused herein, the term “target-binding magnetic particles” refers tomagnetic particles conjugated to target-binding molecules.

The target-binding magnetic particles can be paramagnetic,superparamagnetic, or ferromagnetic. In some embodiments, thetarget-binding magnetic particles can be paramagnetic orsuperparamagnetic. In some embodiments, the target-binding magneticparticles can have the same core magnetic particles as the magneticfield gradient concentrating particles, optionally with differentsurface properties, e.g., surface chemistry. In other embodiments, thecore magnetic particles within the target-binding magnetic particles canbe different from that of the magnetic field gradient concentratingparticles.

The target-binding magnetic particles can range in size from 1 nm to 1mm. For example, the target-binding magnetic particles can be about 2.5nm to about 500 μm, or about 5 nm to about 250 μm in size. In someembodiments, the target-binding magnetic particles can be about 5 nm toabout 100 μm in size. In some embodiments, the target-binding magneticparticles can be about 0.01 μm to about 10 μm in size. In someembodiments, the target-binding magnetic particles can be about 0.05 μmto about 5 μm in size. In some embodiments, the target-binding magneticparticles can be about 0.08 μm to about 1 μm in size. In one embodiment,the target-binding magnetic particles can be about 10 nm to about 10 μmin size. In some embodiments, the target-binding magnetic particles canhave a size ranging from about 1 nm to about 1000 nm, from about 10 nmto about 500 nm, from about 25 nm to about 300 nm, from about 40 nm toabout 250 nm, or from about 50 nm to about 200 nm. In one embodiment,the target-binding magnetic particles can have a size of about 50 nm toabout 200 nm. The target-binding magnetic particles can be manipulatedusing magnetic field or magnetic field gradient. Such particles commonlyconsist of magnetic elements such as iron, nickel and cobalt and theiroxide compounds. Magnetic microbeads are well-known and methods fortheir preparation have been described in the art. See, e.g., U.S. Pat.Nos. 6,878,445; 5,543,158; 5,578,325; 6,676,729; 6,045,925; and7,462,446; and U.S. Patent Publication Nos. 2005/0025971; 2005/0200438;2005/0201941; 2005/0271745; 2006/0228551; 2006/0233712; 2007/01666232;and 2007/0264199, the contents of which are incorporated herein byreference.

The target-binding magnetic particles can be of any shape, including butnot limited to spherical, rod, elliptical, cylindrical, and disc.

Target-binding microparticles: The target-binding microparticlecomprises at least one target-binding molecule on its surface. In someembodiments, the target-binding molecules can be pre-bound with ablocking agent described herein. The term “microparticle” as used hereinrefers to a particle having a particle size of about 0.001 μm to about100 μm, about 0.005 μm to about 50 μm, about 0.01 μm to about 25 μm,about 0.05 μm to about 10 μm, or about 0.05 μm to about 5 μm. In oneembodiment, the microparticle has a particle size of about 0.05 μm toabout 1 μm. In one embodiment, the microparticle is about 0.09 μm-about0.2 μm in size. It will be understood by one of ordinary skill in theart that microparticles usually exhibit a distribution of particle sizesaround the indicated “size.” Unless otherwise stated, the term “size” asused herein refers to the mode of a size distribution of microparticles,i.e., the value that occurs most frequently in the size distribution.Methods for measuring the microparticle size are known to a skilledartisan, e.g., by dynamic light scattering (such as photocorrelationspectroscopy, laser diffraction, low-angle laser light scattering(LALLS), and medium-angle laser light scattering (MALLS)), lightobscuration methods (such as Coulter analysis method), or othertechniques (such as rheology, and light or electron microscopy).

The microparticles can be of any shape, e.g., a sphere. In someembodiments, the term “microparticle” as used herein can encompass amicrosphere. The term “microsphere” as used herein refers to amicroparticle having a substantially spherical form. A substantiallyspherical microparticle is a microparticle with a difference between thesmallest radii and the largest radii generally not greater than about40% of the smaller radii, and more typically less than about 30%, orless than 20%. In one embodiment, the term “microparticle” as usedherein encompasses a microcapsule. The term “microcapsule” as usedherein refers to a microscopic capsule that contains an activeingredient, e.g., a therapeutic agent.

In some embodiments, the microparticles can comprise biocompatiblepolymers as described herein.

In general, any biocompatible material well known in the art forfabrication of microparticles can be used in embodiments of themicroparticle described herein. Accordingly, a microparticle comprisinga lipidic microparticle core is also within the scope described herein.An exemplary lipidic microparticle core is, but is not limited to, aliposome. A liposome is generally defined as a particle comprising oneor more lipid bilayers enclosing an interior, e.g., an aqueous interior.In one embodiment, a liposome can be a vesicle formed by a bilayer lipidmembrane. Methods for the preparation of liposomes are well described inthe art, e.g., Szoka and Papahadjopoulos (1980) Ann. Rev. Biophys.Bioeng. 9: 467, Deamer and Uster (1983) Pp. 27-51 In: Liposomes, ed. M.J. Ostro, Marcel Dekker, New York.

Target-binding microtiter plates: In some embodiments, the bottomsurface of microtiter wells can be coated with the target-bindingmolecules described herein, e.g., for detecting and/or determining theamount of a target entity in a sample. In some embodiments, thetarget-binding molecules can be pre-bound with a blocking agentdescribed herein. After a target entity in the sample displacing theblocking agent and binding to the target-binding molecules bound to themicrowell surface, the rest of the sample can be removed. Detectablemolecules that can also bind to a target entity (e.g., a target-bindingmolecule conjugated to a detectable molecules as described herein) canthen be added to the microwells with the target entity for detection ofthe target entity. Various signal detection methods for determining theamount of proteins, e.g., using enzyme-linked immunosorbent assay(ELISA), with different detectable molecules have been well establishedin the art, and those signal detection methods can also be employedherein to facilitate detection of the signal induced by a target entitybinding on the target-binding molecules.

Target-binding dipsticks/test strips: In some embodiments, thetarget-binding molecules can be adapted for use in a dipstick and/or atest strip for detection of a target entity. For example, a dipstickand/or a test strip can include at least one test area containing one ormore target-binding molecules described herein. In some embodiments, thetarget-binding molecules can be conjugated or attached to a test areasurface of the dipstick and/or a test strip. Methods for conjugating aprotein to a solid substrate surface are known in the art, including,but not limited to direct cross-linking, indirect cross-linking via acoupling agent (e.g., a functional group, a peptide, a nucleic acidmatrix such as DNA matrix), absorption, or any other art-recognizedmethods known in the art.

In some embodiments, the target-binding molecule(s) conjugated to thedipstick and/or a test strip can further comprise a detectable label asdescribed herein. In some embodiments, the target-binding molecules canbe pre-bound with a blocking agent described herein. In someembodiments, the dipstick and/or a test strip can further comprise atleast one reference area or control area for comparison with a readoutsignal determined from the test area. The reference area generallyexcludes the target-binding molecules, e.g., to account for anybackground signal. In some embodiments, the reference area can includeone or more known amounts of the detectable label that thetarget-binding molecules in the test area encompass. In suchembodiments, the reference area can be used for calibration such thatthe amount of the target entity in a test sample can be estimated orquantified.

The dipstick and/or a test strip can be in any shape and/or in anyformat, e.g., a planar shape such as a rectangular strip or a circulardisk, or a curved surface such as a stick. Alternatively, a continuousroll can be utilized, rather than discrete test strips, on which thetest area(s) and optionally reference area(s) are present in the form ofcontinuous lines or a series of spots.

The dipstick and/or a test strip can be made of any material, including,without limitations, paper, nitrocellulose, glass, plastic, polymer,membrane material, nylon, and any combinations thereof. In oneembodiment, the dipstick and/or a test strip can include paper. In oneembodiment, the dipstick and/or a test strip can include nylon.

The target-binding dipsticks and/or test strips described herein can beused as point-of-care diagnostic tools for detection of specific targetentity such as microbes. By way of example only, a microbe-bindingdipstick or test strip (e.g., made of membrane material such as nylon)comprising microbe-binding molecules and a blocking agent bound theretocan be brought into contact with a test sample (e.g., a blood sample)from a patient or a subject, and incubated for a period of time, e.g.,at least about 15 seconds, at least about 30 seconds, at least about 1min, at least about 2 mins, at least about 5 mins, at least about 10mins, at least about 15 mins, at least about 30 mins, at least about 1hour or more. Depending on different embodiments of the target-bindingmolecules, in some embodiments, the microbe-binding dipstick or teststrip after contact with a test sample (e.g., a blood sample) can befurther contacted with at least one additional agent to facilitatedetection of microbes, and/or to increase specificity of the microbedetection. For example, some embodiments of the dipstick or test stripafter contact with a test sample (e.g., a blood sample) can be furthercontacted with a detectable label that is conjugated to a molecule thatbinds to a microbe and/or microbial matter. Examples of such moleculescan include, but are not limited to, one or more embodiments of thetarget-binding molecule described herein, an antibody specific for themicrobes or pathogens to be detected, a protein, a peptide, acarbohydrate or a nucleic acid that is recognized by the microbes orpathogens to be detected, and any combinations thereof.

In some embodiments, the target-binding agent or substrates can beconfigured to detect and/or capture at least one microbe and/or afragement thereof. Thus, in some embodiments, the target-binding agentor substrate can comprise a microbe-binding agent. By way of exampleonly, a microbe-binding agent can comprise a carbohydrate recognitiondomain of a lectin (e.g., FcMBL), and/or any other microbe-bindingmolecules described in WO/2011/090954 (corresponding U.S. patentapplication Ser. No. 13/574,191 entitled “Engineered opsonin forpathogen detection and treatment”) and WO/2013/012924 (correspondingU.S. patent application Ser. No. 14/233,553 entitled “Engineeredmicrobe-targeting molecules and uses thereof”) and the contents of whichare incorporated herein by reference.

Target-binding molecules: The target-binding agent can be present in anyform, including but not limited to a target-binding molecule, and/or atarget-binding substrate (e.g., a target-binding molecule conjugated toa solid substrate) as described above. By “target-binding molecules” ismeant herein molecules that can interact with or bind to a targetspecies or a target analyte such that the target species or targetanalyte can be captured or isolated from a fluid sample. Typically thenature of the interaction or binding is noncovalent, e.g., by hydrogen,electrostatic, or van der Waals interactions, however, binding can alsobe covalent. Target-binding molecules can be naturally-occurring,recombinant or synthetic. Examples of the target-binding molecule caninclude, but are not limited to, a nucleic acid, an antibody or aportion thereof, an antibody-like molecule, an enzyme, an antigen, asmall molecule, a protein, a peptide, a peptidomimetic, a carbohydrate,an aptamer, and any combinations thereof. By way of example only, inimmunohistochemistry, the target-binding molecule can be an antibodyspecific to the target antigen to be analyzed. An ordinary artisan canreadily identify appropriate target-binding molecules for each targetspecies or analytes of interest to be detected in various bioassays.

In some embodiments, the target-binding molecules can be pre-bound witha blocking agent described herein.

In some embodiments, the target-binding molecules can be modified by anymeans known to one of ordinary skill in the art. Methods to modify eachtype of target-binding molecules are well recognized in the art.Depending on the types of target-binding molecules, an exemplarymodification includes, but is not limited to genetic modification,biotinylation, labeling (for detection purposes), chemical modification(e.g., to produce derivatives or fragments of the target-bindingmolecule), and any combinations thereof. In some embodiments, thetarget-binding molecule can be genetically modified. In someembodiments, the target-binding molecule can be biotinylated.

In some embodiments, the target-binding molecules can comprise on theirsurfaces microbe-binding molecules as described herein, and/or disclosedin WO/2011/090954 (corresponding U.S. patent application Ser. No.13/574,191 entitled “Engineered opsonin for pathogen detection andtreatment”) and WO/2013/012924 (corresponding U.S. patent applicationSer. No. 14/233,553 entitled “Engineered microbe-targeting molecules anduses thereof”), the contents of which are incorporated herein byreference. Accordingly, in some embodiments, the method described hereincan be used with the target-binding magnetic particles for microbialcapture, i.e., microbe-binding magnetic particles, e.g., but not limitedto FcMBL magnetic particles. Examples of microbe-binding magneticparticles can include, but are not limited to the ones described inWO/2011/090954 (corresponding U.S. patent application Ser. No.13/574,191 entitled “Engineered opsonin for pathogen detection andtreatment”) and WO/2013/012924 (corresponding U.S. patent applicationSer. No. 14/233,553 entitled “Engineered microbe-targeting molecules anduses thereof”), the contents of which are incorporated herein byreference.

In some embodiments, the target-binding molecule can be an antibody or aportion thereof, or an antibody-like molecule. In some embodiments, thetarget-binding molecule can be an antibody or a portion thereof, or anantibody-like molecule that is specific for detection of a rare-cell,e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe biomarker. In some embodiments, the target-binding molecule canbe an antibody or a portion thereof, or an antibody-like molecule thatis specific for a protein or an antigen present on the surface of a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe. In such embodiments, the target-binding molecules can be usedto, for example, detect and/or identify cell type or species (includingnormal and/or diseased cells), the presence of cell or disease markers,cellular protein expression levels, phosphorylation or otherpost-translation modification state, or any combinations thereof.

In some embodiments, the target-binding molecule can be a nucleic acid(e.g., DNA, RNA, LNA, PNA, or any combinations thereof). For example,the nucleic acid can encode the gene specific for a rare cell biomarker,e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe biomarker. In such embodiments, the nucleic acids can be used todetermine, for example, the existence of characteristic cellular DNA orRNA sequences (such as in fluorescent in situ hybridization), RNAexpression levels, miRNA presence and expression, and any combinationsthereof, in various applications, e.g., for disease diagnose, prognosisand/or monitoring.

In some embodiments, the target-binding molecule can be a protein or apeptide. In some embodiments, the protein or peptide can be essentiallyany proteins that can bind to a rare cell, e.g., a circulating tumorcell, a fetal cell, a stem cell and/or a microbe. By way of exampleonly, if the target species is a bacteria, exemplary proteins orpeptides that can be used to generate microbe-binding molecules and/ormicrobe-binding magnetic particles can include, but are not limited to,innate-immune proteins (e.g., without limitations, MBL, Dectin-1, TLR2,and TLR4 and any molecules (including recombinant or engineered proteinmolecules) disclosed here as well as the microbe-binding moleculesdisclosed in the International Application Publication Nos.WO/2011/090954 and WO/2013/012924, the content of which is incorporatedherein by reference) and proteins comprising the chitin-binding domain,and any factions thereof. Such innate-immune proteins and chitin-bindingdomain proteins can be used to detect their correspondingpattern-recognition targets (e.g., microbes such as bacteria) andfungus, respectively.

In some embodiments, the target-binding molecule can be an aptamer. Insome embodiments, the target-binding molecule can be a DNA or RNAaptamer. The aptamers can be used in various bioassays, e.g., in thesame way as antibodies or nucleic acids described herein. For example,the DNA or RNA aptamer can encode a nucleic acid sequence correspondingto a rare cell biomarker or a fraction thereof, for use as atarget-binding molecule on the magnetic particles described herein.

In some embodiments, the target-binding molecule can be a cell surfacereceptor ligand. As used herein, a “cell surface receptor ligand” refersto a molecule that can bind to the outer surface of a cell. Exemplarycell surface receptor ligand includes, for example, a cell surfacereceptor binding peptide, a cell surface receptor binding glycopeptide,a cell surface receptor binding protein, a cell surface receptor bindingglycoprotein, a cell surface receptor binding organic compound, and acell surface receptor binding drug. Additional cell surface receptorligands include, but are not limited to, cytokines, growth factors,hormones, antibodies, and angiogenic factors. In some embodiments, anyart-recognized cell surface receptor ligand that can bind to a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe, can be used as a target-binding molecule on the magneticparticles described herein.

Compositions Comprising a Target-Binding Agent (or a Target-BindingSubstrate) and a Blocking Agent

Various embodiments of the methods described herein can be adapted tovarious applications or be integrated as part of a process, e.g., butnot limited to, antibody-based assays (e.g., ELISA), filtrations,microbe detection and/or capture, antibiotic susceptibility testings,multiplexing assays, coating processes, hybridization-based assays,diagnostic strips, targeted drug delivery, or any combinations thereof.Thus, various compositions comprising a target-binding agent and atleast one blocking agent can be formulated to suit the need of eachindividual application. Accordingly, another aspect provided hereinrelates to a composition comprising one or more embodiments of atarget-binding agent described herein, and at least one embodiment of ablocking agent described herein at a pre-determined concentration. Insome embodiments, a composition can comprise one or more embodiments ofa target-binding substrate described herein and at least one embodimentof a blocking agent described herein at a pre-determined concentration.The effective binding affinity of said at least one blocking agent forthe target-binding agent is lower than the effective binding affinity ofa target entity to be captured, and wherein the effective bindingaffinity of said at least one blocking agent for the target-bindingagent is higher than the effective binding affinity of at least oneinterfering molecule present in a sample to be assayed for thetarget-binding agent.

In some embodiments, said at least one blocking agent can be pre-boundto the target-binding agent within the composition. In some embodiments,said at least one blocking agent can be pre-bound to the target-bindingmolecules present on the target-binding substrates. In alternativeembodiments, said at least one blocking agent and the target-bindingagent (or the target-binding substrate) can be kept separately withinthe composition, e.g., each is contained in a separate container. Insome embodiments, the target-binding agent and said at least oneblocking agent can each be independently present in a buffered solution.

In some embodiments, the target-binding agent can comprise an antibody(e.g., a primary antibody, and/or a secondary antibody). In theseembodiments, by way of example only, the composition can be used duringimmunoglobulin secondary detection reactions, immunostaining, and/orELISA assay.

In one embodiment, the addition of a blocking agent to a test sample ina competitive manner can be used in immunoglobulin secondary detectionreactions. An example of such an application is described below:Fluorescent-labeled IgG1 has been raised to detect rabbit F(c) fragmentfor which IgG1 has high affinity. However, IgG1 also has a low affinityto goat F(c) and a medium affinity to an aptamer derived from the rabbitF(c) epitope. Thus, incubating HRP-labeled IgG1 in multiplex labelingassay where a goat primary Ab and a rabbit primary Ab are both used canresult in the fluorescent labeling of both the goat and rabbit primaryAbs.

In order to distinguish the labeling of both the goat and rabbit primaryAbs, fluorescent-labeled IgG1 can be incubated with the aptamers(derived from the rabbit F(c) epitope) with a medium affinity prior toaddition into a multiplex labeling assay where both a goat primary Aband a rabbit are used. In this example, the high affinity ligand A isthe rabbit Ab, the low affinity undesirable ligand B is the goat Ab andthe intermediate affinity ligand C is the aptamer. The rabbit Ab (A) candisplace the aptamers (C) that are bound to the fluorescent-labeled IgG1but the goat Ab (B) is less likely to bind to the fluorescent-labeledIgG1 because the fluorescent-labeled IgG1 has already bound to theaptamers, which cannot be displaced by the goat Ab (B) with loweraffinity. In some embodiments, the goat Ab (B) that is not bound to thefirst fluorescent-labeled IgG1 can be then detected with anotherfluorescent-labeled IgG1, thus enabling detection of different targetentities in a multiplex labeling assay, e.g., using the same detectionagent (e.g., IgG1) but with a different detectable label (e.g., adifferent fluorescent label) for each target entity (e.g., rabbit Ab andgoat Ab).

In some embodiments, the target-binding agent can comprise amicrobe-binding agent (e.g., FcMBL molecule). Examples ofmicrobe-binding agent for detection and/or capture of microbes and/orfragments thereof are known in the art, including, e.g., microbe-bindingmolecules disclosed herein and in the International Application Nos.WO/2011/090954 (corresponding U.S. patent application Ser. No.13/574,191 entitled “Engineered opsonin for pathogen detection andtreatment”) and WO/2013/012924 (corresponding U.S. patent applicationSer. No. 14/233,553 entitled “Engineered microbe-targeting molecules anduses thereof”), the contents of which are incorporated herein byreference.

In some embodiments where the microbe-binding agent comprises amannan-binding domain (e.g., FcMBL molecule), said at least one blockingagent can comprise glucose, maltose, N-acetyl muramic acid, and/or anycombinations thereof. The pre-determined concentration of the blockingagent can vary with the binding affinity of the target entity for thetarget-binding agent. For example, a higher concentration of theblocking agent can be used without adversely affect the binding of thetarget-binding agent to the target entity with a higher effectivebinding affinity. In some embodiments where the glucose is the blockingagent, the pre-determined concentration of glucose can range from about5 mM to about 200 mM.

In some embodiments, the blocking agent can further comprise adetectable label as described herein.

Kits Comprising a Composition Described Herein

A kit comprising at least one composition described herein is alsoprovided. In some embodiments, the kit comprises a first compositioncomprising a first target-binding agent and at least one first blockingagent at a first pre-determined concentration, wherein the effectivebinding affinity of said at least one first blocking agent for the firsttarget-binding agent is lower than the effective binding affinity of afirst target entity to be captured, and wherein the effective bindingaffinity of said at least one first blocking agent for the firsttarget-binding agent is higher than the effective binding affinity of atleast one first interfering molecule present in a sample to be assayedfor the first target-binding agent; and instructions for using thecomposition for detecting or capturing the first target entity.

In some embodiments, the kit can further comprise a second compositioncomprising a second target-binding agent. In some embodiments, the kitcan further comprise at least one second blocking agent. The secondblocking agent can be included in the kit at a second pre-determinedconcentration. In some embodiments, the effective binding affinity of asecond blocking agent for the second target-binding agent can be lowerthan the effective binding affinity of a second target entity to becaptured, and the effective binding affinity of the second blockingagent for the second target-binding agent can be higher than theeffective binding affinity of at least one second interfering moleculepresent in the sample to be assayed for the second target-binding agent.

In some embodiments, the first blocking agent and the second blockingagent can be selected to prevent or reduce the binding of the sameinterfering agent to the first target-binding agent and the secondtarget-binding agent, respectively. By way of example only, where saidat least the first interfering agent and/or said at least the secondinterfering agent can be a non-specific binding molecule present in asample, the first blocking agent and the second blocking agent can beselected to prevent or reduce the binding of non-specific bindingmolecules to the first target-binding agent and the secondtarget-binding agent, respectively.

In some embodiments, the first blocking agent and the second blockingagent can be selected to prevent or reduce the binding of a differentinterfering agent to the first target-binding agent and the secondtarget-binding agent, respectively. By way of example only, where thekit is adapted for use in a multiplexing assay, a first target entitycan be intended to be detected by a first target-binding agent but not asecond target-binding agent, while a second target entity can beintended to be detected by a second target-binding agent, but not afirst target-binding agent. In these embodiments, the first targetentity can be considered as said second interfering agent with respectto binding interaction between the second target-binding agent and thesecond target entity, and the second target entity can be considered assaid first interfering agent with respect to binding interaction betweenthe first target-binding agent and the first target entity. As usedherein, the term “multiplexing” refers to simultaneous detection of morethan one target entity, e.g., at least 2 target entities, at least 3target entities, at least 4 target entities, or more.

In some embodiments, the first blocking agent can be pre-bound to thefirst target-binding agent. In some embodiments, the second blockingagent can be pre-bound to the second target-binding agent.

In some embodiments, the first target-binding agent can be affixed to afirst solid substrate. In some embodiments, the first solid substratecan be further affixed with the second target-binding agent. In someembodiments, the second target-binding agent can be affixed to a secondsolid substrate. Non-limiting examples of the first or the second solidsubstrate includes, but are not limited to, a nucleic acid scaffold, aprotein scaffold, a lipid scaffold, a dendrimer, microparticle or amicrobead, a nanotube, a microtiter plate, a medical apparatus orimplant, a microchip, a filtration device, a membrane, a diagnosticstrip, a dipstick, an extracorporeal device, a mixing element (e.g., aspiral mixer), a microscopic slide, a hollow fiber, a hollow fibercartridge, and any combination thereof.

In some embodiments, the kit can further comprise a first detectionagent capable of binding to the first target entity. In someembodiments, the kit can further comprise a second detection agentcapable of binding to the second target entity.

In addition to the above mentioned components, any embodiments of thekits described herein can include informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the aggregates for the methods described herein. For example, theinformational material can describe methods for using the kits providedherein to perform an assay for capture and/or detection of a targetentity, e.g., a microbe. The kit can also include an empty containerand/or a delivery device, e.g., which can be used to deliver a testsample to a test container.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments, the kit can contain separate containers, dividersor compartments for each component and informational material. Forexample, each different component can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, acollection of the magnetic particles is contained in a bottle, vial orsyringe that has attached thereto the informational material in the formof a label.

Test Sample or Sample

In accordance with various embodiments described herein, a test sample,including any fluid or specimen (processed or unprocessed) that isintended to be evaluated for the presence of a target entity can besubjected to methods, compositions, kits and systems described herein.The test sample or fluid can be liquid, supercritical fluid, solutions,suspensions, gases, gels, slurries, and combinations thereof. The testsample or fluid can be aqueous or non-aqueous.

In some embodiments, the test sample can be an aqueous fluid. As usedherein, the term “aqueous fluid” refers to any flowable water-containingmaterial that is suspected of comprising a pathogen.

In some embodiments, the test sample can include a biological fluidobtained from a subject. Exemplary biological fluids obtained from asubject can include, but are not limited to, blood (including wholeblood, plasma, cord blood and serum), lactation products (e.g., milk),amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid,bronchial aspirate, perspiration, mucus, liquefied stool sample,synovial fluid, lymphatic fluid, tears, tracheal aspirate, and anymixtures thereof. In some embodiments, a biological fluid can include ahomogenate of a tissue specimen (e.g., biopsy) from a subject. In oneembodiment, a test sample can comprises a suspension obtained fromhomogenization of a solid sample obtained from a solid organ or afragment thereof.

In some embodiments, the test sample can be a whole blood sampleobtained from a subject suspected of having a microbe infection (e.g., apathogen infection).

In some embodiments, the test sample can include a fluid or specimenobtained from an environmental source. For example, the fluid orspecimen obtained from the environmental source can be obtained orderived from food products or industrial food products, food produce,poultry, meat, fish, beverages, dairy products, water (includingwastewater), surfaces, ponds, rivers, reservoirs, swimming pools, soils,food processing and/or packaging plants, agricultural places,hydrocultures (including hydroponic food farms), pharmaceuticalmanufacturing plants, animal colony facilities, and any combinationsthereof.

In some embodiments, the test sample can include a fluid or specimencollected or derived from a biological culture. For example, abiological culture can be a cell culture. Examples of a fluid orspecimen collected or derived from a biological culture includes the oneobtained from culturing or fermentation, for example, of single- ormulti-cell organisms, including prokaryotes (e.g., bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, fungi), andincluding fractions thereof. In some embodiments, the test sample caninclude a fluid from a blood culture. In some embodiments, the culturemedium can be obtained from any source, e.g., without limitations,research laboratories, pharmaceutical manufacturing plants,hydrocultures (e.g., hydroponic food farms), diagnostic testingfacilities, clinical settings, and any combinations thereof.

In some embodiments, the test sample can be a fluid or specimencollected or derived from a microbe colony.

In some embodiments, the test sample can include a media or reagentsolution used in a laboratory or clinical setting, such as forbiomedical and molecular biology applications. As used herein, the term“media” refers to a medium for maintaining a tissue, an organism, or acell population, or refers to a medium for culturing a tissue, anorganism, or a cell population, which contains nutrients that maintainviability of the tissue, organism, or cell population, and supportproliferation and growth.

As used herein, the term “reagent” refers to any solution used in alaboratory or clinical setting for biomedical and molecular biologyapplications. Reagents include, but are not limited to, salinesolutions, PBS solutions, buffered solutions, such as phosphate buffers,EDTA, Tris solutions, and any combinations thereof. Reagent solutionscan be used to create other reagent solutions. For example, Trissolutions and EDTA solutions are combined in specific ratios to create“TE” reagents for use in molecular biology applications.

In some embodiments, the test sample can be a non-biological fluid. Asused herein, the term “non-biological fluid” refers to any fluid that isnot a biological fluid as the term is defined herein. Exemplarynon-biological fluids include, but are not limited to, water, saltwater, brine, buffered solutions, saline solutions, sugar solutions,carbohydrate solutions, lipid solutions, nucleic acid solutions,hydrocarbons (e.g. liquid hydrocarbons), acids, gasolines, petroleum,liquefied samples (e.g., liquefied samples), and mixtures thereof

Capture and/or Detection of a Microbe and/or Microbial Fragments/Matterin a Test Sample

In some embodiments, the methods, compositions and/or kits describedherein can be used for capture and/or detection a microbe and/ormicrobial fragments/matter in a test sample. Specifically, in someembodiments, the microbe-binding agents (e.g., FcMBL-coated magneticparticles or FcMBL-coated hollow fibers) can be pretreated with at leastone blocking agent as described herein, prior to contacting a testsample with the microbe-binding agents (e.g., FcMBL-coated magneticparticles or FcMBL-coated hollow fibers). The blocking agent is selectedto have an effective binding affinity for the microbe-binding agents(e.g., FcMBL-coated magnetic particles or FcMBL-coated hollow fibers)that is lower than the effective binding affinity of the microbes and/orfragments thereof for the microbe-binding agents (e.g., FcMBL-coatedmagnetic particles or FcMBL-coated hollow fibers); and wherein theblocking agent is selected to have the effective binding affinity forthe microbe-binding agents (e.g., FcMBL-coated magnetic particles orFcMBL-coated hollow fibers) that is higher than the effective bindingaffinity of at least one interfering agent present in the sample for themicrobe-binding agents (e.g., FcMBL-coated magnetic particles orFcMBL-coated hollow fibers). In these embodiments, the microbes orfragments thereof can displace the blocking agent bound to themicrobe-binding agents (e.g., FcMBL-coated magnetic particles orFcMBL-coated hollow fibers) and become captured on the microbe-bindingagents (e.g., FcMBL-coated magnetic particles or FcMBL-coated hollowfibers).

The high affinity binding of the captured microorganism can displace theblocking agent (e.g., sugar such as glucose) and prevent undesirablebinding of blood cells and other non-target materials that wouldinterfere with downstream detection/analysis processes, e.g., ATP-baseddetection of viable bacteria or downstream genetic amplificationefficiency or immunoenzymatic detection. In one embodiment, whenperforming an assay using a dialysis-like therapeutic (DLT) device,e.g., as described in the International Application Publication No.WO/2012/135834, the content of which is incorporated herein byreference, FcMBL beads or membrane can be preloaded with a blockingagent (e.g., but not limited to, glucose and/or maltose) to not onlyenhance the capture of pathogens and microbial carbohydrate compoundsbut also to enhance magnetic bead recovery and prevent FcMBLinactivation by low affinity binders such as erythrocytes.

An example process for capture of a microbe and/or fragments thereof ina test sample comprises contacting a test sample with a microbe-bindingagent. The microbe-binding agent can be pre-bound with a blocking agentas described herein, or added concurrently with a blocking agent to atest sample. After allowing microbes and/or fragments thereof, ifpresent, to bind to the microbe-binding agent, e.g., by displacing theblocking agent from the microbe-binding agent, the microbe-binding agentcomprising microbes and/or fragments thereof bound thereto can beseparated from the test sample. In some embodiments, the microbes and/orfragments thereof can be detected and/or identified.

An exemplary process for detecting a microbe (e.g., a pathogen) and/ormicrobial fragment/matter in a test sample is shown in FIG. 5. Theprocess is provided for illustration purpose only, and one or more stepscan be added, deleted, substituted and/or combined together. As shown inFIG. 5, the process 1200 comprises the optional step 1202 (preprocessingof the sample), step 1204 (processing of the sample), step 1206comprising 1208 (microbe capture, e.g., pathogen capture) and 1210(microbe separation, e.g., pathogen separation), step 1212 (detection ofmicrobe and/or microbe identity and number), the optional step 1213(microbe and/or detection agent immobilization), and the optional stepsfor antibiotic susceptibility testing, if desired, comprising step 1214(incubation with the antibiotic agent) and step 1216 (detection ofmicrobe number and/or viability). While these are discussed as discreteprocesses, one or more of the preprocessing, processing, capture,microbe separation, detection, and antibiotic sensitivity can beperformed in a system. In one embodiment, one or more of thepreprocessing, processing, capture, microbe separation, detection, andantibiotic sensitivity can be performed in a microfluidic device. Insome embodiments, one or more of the microbe capture or separation,microbe incubation, and microbe detection can be included in amicrofluidic device. In some embodiments, one or more of the modules orsystems performing microbe capture or separation, microbe incubation,and microbe detection can comprise a microfluidic channel. Use of amicrofluidic device can automate the process and/or allow processing ofmultiple samples at the same time. One of skill in the art is well awareof methods in the art for collecting, handling and processing biologicalfluids which can be used in the practice of the present disclosure.Additionally, the microfluidic devices for the various steps can becombined into one system for carrying out the method described herein.For example, such a system can comprise two or more of the following:(i) a capture or separation system for capturing a microbe from abiological fluid; (ii) an incubation system for incubating the microbewith or without an antibiotic agent; and (iii) a detection system fordetecting the microbe after incubation. Alternatively, the various stepscan also be carried out using separate systems or devices.

1202 (Sample preprocessing): It can be necessary or desired that a testsample, such be preprocessed prior to microbe detection as describedherein, e.g., with a preprocessing reagent. Even in cases wherepretreatment is not necessary, preprocess optionally can be done formere convenience (e.g., as part of a regimen on a commercial platform).A preprocessing reagent can be any reagent appropriate for use with themethods described herein.

The sample preprocessing step generally comprises adding one or morereagents to the sample. This preprocessing can serve a number ofdifferent purposes, including, but not limited to, hemolyzing cells suchas blood cells, dilution of sample, etc. The preprocessing reagents canbe present in the sample container before sample is added to the samplecontainer or the preprocessing reagents can be added to a sample alreadypresent in the sample container. When the sample is a biological fluid,the sample container can be a VACUTAINER®, e.g., a heparinizedVACUTAINER®.

The preprocessing reagents include, but are not limited to, surfactantsand detergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases and the like), and solvents, such as buffersolutions.

In some embodiments, a preprocessing reagent is a surfactant or adetergent. In one embodiment, the preprocessing reagent is Triton X100.

After addition of the preprocessing reagent, the reagent can be mixedinto the sample. This can be simply accomplished by agitating thesample, e.g., shaking the sample and/or moving the sample around in amicrofluidic device.

1204 (Sample processing): After the optional preprocessing step, thesample can be optionally further processed by adding one or moreprocessing reagents to the sample. These processing reagents can degradeunwanted molecules present in the sample and/or dilute the sample forfurther processing. These processing reagents include, but are notlimited to, surfactants and detergents, salts, cell lysing reagents,anticoagulants, degradative enzymes (e.g., proteases, lipases,nucleases, lipase, collagenase, cellulases, amylases, heparanases, andthe like), and solvents, such as buffer solutions. Amount of theprocessing reagent to be added can depend on the particular sample to beanalyzed, the time required for the sample analysis, identity of themicrobe to be detected or the amount of microbe present in the sample tobe analyzed.

It is not necessary, but if one or more reagents are to be added theycan present in a mixture (e.g., in a solution, “processing buffer”) inthe appropriate concentrations. Amount of the various components of theprocessing buffer can vary depending upon the sample, microbe to bedetected, concentration of the microbe in the sample, or time limitationfor analysis.

Generally, addition of the processing buffer can increase the volume ofthe sample by 5%, 10%, 15%, 20% or more. In some embodiments, about 50μl to about 500 μl of the processing buffer are added for each ml of thesample. In some embodiments, about 100 μl to about 250 μl of theprocessing buffer are added for each ml of the sample. In oneembodiment, about 125 μl of the processing buffer are added for each mlof the sample.

In some embodiments, a detergent or surfactant comprises about 5% toabout 20% of the processing buffer volume. In some embodiment, adetergent or surfactant comprises about 5% to about 15% of theprocessing buffer volume. In one embodiment, a detergent or surfactantcomprises about 10% of the processing buffer volume.

In some embodiments, one ml of the processing buffer comprises about 1 Uto about 100 U of a degradative enzyme. In some embodiments, one ml ofthe processing buffer comprises about 5 U to about 50 U of a degradativeenzyme. In one embodiment, one ml of the processing buffer comprisesabout 10 U of a degradative enzyme. Enzyme unit (U) is an art known termfor the amount of a particular enzyme that catalyzes the conversion of 1μmmol of substrate per minute.

In some embodiments, one ml of the processing buffer comprises about 1μg to about 10 μg of an anti-coagulant. In some embodiment, one ml ofthe processing buffer comprises about 1 μg to about 5 μg of ananti-coagulant. In one embodiment, one ml of the processing buffercomprises about 4.6 μg of an anti-coagulant.

In some embodiments, one ml of the processing buffer comprises about 1mg to about 10 mg of anti-coagulant. In some embodiment, one ml of theprocessing buffer comprises about 1 mg to about 5 mg of anti-coagulant.In one embodiment, one ml of the processing buffer comprises about 4.6mg of anti-coagulant.

Exemplary anti-coagulants include, but are not limited to, heparin,heparin substitutes, salicylic acid, D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone (PPACK), Hirudin, ANCROD® (snake venom, VIPRONAX®),tissue plasminogen activator (tPA), urokinase, streptokinase, plasmin,prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors,dextrans, thrombin antagonists/inhibitors, ethylene diamine tetraaceticacid (EDTA), acid citrate dextrose (ACD), sodium citrate, citratephosphate dextrose (CPD), sodium fluoride, sodium oxalate, sodiumpolyanethol sulfonate (SPS), potassium oxalate, lithium oxalate, sodiumiodoacetate, lithium iodoacetate and mixtures thereof.

Suitable heparinic anticoagulants include heparins or active fragmentsand fractions thereof from natural, synthetic, or biosynthetic sources.Examples of heparin and heparin substitutes include, but are not limitedto, heparin calcium, such as calciparin; heparin low-molecular weight,such as enoxaparin (lovenox®), Bemiparin, Certoparin, Dalteparin,Nadroparin, Parnaparin, Reviparin or Tinzaparin; heparin sodium, such asheparin, lipo-hepin, liquaemin sodium, and panheprin; heparin sodiumdihydroergotamine mesylate; lithium heparin; and ammonium heparin.

Suitable prothrombopenic anticoagulants include, but are not limited to,anisindione, dicumarol, warfarin sodium, and the like.

Examples of phosphodiesterase inhibitors suitable for use in the methodsdescribed herein include, but are not limited to, anagrelide,dipyridamole, pentoxifyllin, and theophylline.

Suitable dextrans include, but are not limited to, dextran70, such asHYSKONTM (CooperSurgical, Inc., Shelton, Conn., U.S.A.) and MACRODEXTM(Pharmalink, Inc., Upplands Vasby, Sweden), and dextran 75, such asGENTRANTM 75 (Baxter Healthcare Corporation).

Suitable thrombin antagonists include, but are not limited to, hirudin,bivalirudin, lepirudin, desirudin, argatroban, melagatran, ximelagatranand dabigatran.

As used herein, anticoagulants can also include factor Xa inhibitors,factor Ha inhibitors, and mixtures thereof. Various direct factor Xainhibitors are known in the art including, those described in Hirsh andWeitz, Lancet, 93:203-241, (1999); Nagahara et al. Drugs of the Future,20: 564-566, (1995); Pinto et al, 44: 566-578, (2001); Pruitt et al,Biorg. Med. Chem. Lett., 10: 685-689, (2000); Quan et al, J. Med. Chem.42: 2752-2759, (1999); Sato et al, Eur. J. Pharmacol, 347: 231-236,(1998); Wong et al, J. Pharmacol. Exp. Therapy, 292:351-357, (2000).Exemplary factor Xa inhibitors include, but are not limited to,DX-9065a, RPR-120844, BX-807834 and SEL series Xa inhibitors. DX-9065ais a synthetic, non-peptide, propanoic acid derivative, 571 D selectivefactor Xa inhibitor. It directly inhibits factor Xa in a competitivemanner with an inhibition constant in the nanomolar range. See forexample, Herbert et al, J. Pharmacol. Exp. Ther. 276:1030-1038 (1996)and Nagahara et al, Eur. J. Med. Chem. 30(suppl):140s-143s (1995). As anon-peptide, synthetic factor Xa inhibitor, RPR-120844 (Rhone-PoulencRorer), is one of a series of novel inhibitors which incorporate3-(S)-amino-2-pyrrolidinone as a central template. The SEL series ofnovel factor Xa inhibitors (SEL1915, SEL-2219, SEL-2489, SEL-2711:Selectide) are pentapeptides based on L-amino acids produced bycombinatorial chemistry. They are highly selective for factor Xa andpotency in the pM range.

Factor Ha inhibitors include DUP714, hirulog, hirudin, melgatran andcombinations thereof. Melagatran, the active form of pro-drugximelagatran as described in Hirsh and Weitz, Lancet, 93:203-241, (1999)and Fareed et al. Current Opinion in Cardiovascular, pulmonary and renalinvestigational drugs, 1:40-55, (1999).

Generally, salt concentration of the processing buffer can range fromabout 10 mM to about 100 mM. In some embodiments, the processing buffercomprises a salt at a concentration of about 25 mM to about 75 mM. Insome embodiment, the processing buffer comprises a salt at aconcentration of about 45 mM to about 55 mM. In one embodiment, theprocessing buffer comprises a salt at a concentration of about 43 mM toabout 45 mM.

The processing buffer can be made in any suitable buffer solution knownto a skilled artisan. In some embodiments, the buffer solution isphysiologically compatible to cells. Alternatively, the processingbuffer can be made in water.

In some embodiments, the processing buffer comprises a mixture ofTriton-X, DNAse I, human plasmin, CaCl₂ and Polysorbate 20. In oneembodiment, the processing buffer consists of a mixture of Triton-X,DNAse I, human plasmin, CaCl₂ and Polysorbate 20 in a TBS buffer.

In one embodiment, one ml of the processing buffer comprises 100 μl ofTriton-X100, 10 μl of DNAse (1 U/1 μl), 10 μl of human plasmin at 4.6mg/ml and 870 μl of a mixture of TBS, 0.1% Polysorbate 20 and 50 mMCaCl₂.

Reagents and treatments for processing blood before assaying are alsowell known in the art, e.g., as used for assays on Abbott TDx, AxSYM®,and ARCHITECT® analyzers (Abbott Laboratories), as described in theliterature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal AntibodyAssay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem.36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYMCyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMITCyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or ascommercially available. Additionally, pretreatment can be done asdescribed in U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0 471 293,U.S. Provisional Pat. App. 60/878,017, filed Dec. 29, 2006, and U.S.Pat. App. Pub. No. 2008/0020401, content of all of which is incorporatedherein by reference. It is to be understood that one or more of theseknown reagents and/or treatments can be used in addition to oralternatively to the sample treatment described herein.

In some embodiments, after addition of the processing buffer, the samplecomprises 1% Triton-X, 10 U of DNase, 4.6 mg/ml of plasmin, 5 mMCalcium, 0.01% of Polysorbate 20, 2.5 mM of Tris, 150 mM of NaCl and 0.2mM of KCl in addition to the components already present in the sample.

After addition of the processing buffer, the sample can undergo mixing.This can be simply accomplished by agitating the sample, e.g., shakingthe sample or moving the sample around in a microfluidic device.

After addition of the processing reagents, the sample can be incubatedfor a period of time, e.g., for at least one minute, at least twominutes, at least three minutes, at least four minutes, at least fiveminutes, at least ten minutes, at least fifteen minutes, at least thirtyminutes, at least forty-five minutes, or at least one hour. Suchincubation can be at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature.

1206 (1208 (microbe capture) and/or 1210 (microbe separation)): Afterprocessing of the sample, the sample can be subjected to a microbecapture process (step 1208). The microbe capture process can allow forconcentrating and/or cleaning up the sample before proceeding withincubation with an antibiotic agent. Without limitations, any methodknown in the art for capturing or extracting or concentrating microbesfrom a biological sample (e.g., a biological fluid) can be used. Asample comprising the extracted microbes from the biological fluid isalso referred to as a microbe sample herein.

The extraction and concentration process can be completed in less than 6hours, less than 5 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 1 hour, less than 30 minutes, less than 15minutes, less than 10 minutes, or shorter. In some embodiments,extraction and concentration of a microbe in the sample can be donewithin 10 minutes to 60 minutes of starting the process. In someembodiments, extraction and concentration of a microbe in the sample canbe done in about 10 minutes, e.g., mixing a sample comprising a microbeto be extracted with at least one microbe-binding substrate (e.g., aplurality of microbe-binding magnetic particles described herein)followed by separation of the microbe-bound microbe-binding substratefrom the rest of the sample.

Additionally, the extraction and concentration process described hereincan be utilized to extract a microbe in a sample of any given volume. Insome embodiments, sample volume is about 0.25 ml to about 50 ml, about0.5 ml to about 25 ml, about 1 ml to about 15 ml, about 2 ml to about 10ml. In some embodiments, sample volume is about 5 ml. In one embodiment,sample volume is 8 ml.

Generally, microbe capturing and isolating or separating microbes fromthe test sample comprises contacting the test sample (e.g., thebiological fluid) with a microbe-binding molecule linked to a solidsubstrate or scaffold (e.g., beads, fibers, filters, beads, filters,fibers, screens, mesh, tubes, hollow fibers, fluidic channels,microfluidic channels, and the like) for capturing and isolating orseparating microbes from the biological fluid.

The microbe capture process comprises mixing a solid substrate, surfaceof which is coated with microbe-binding molecules which can bind to amicrobe in the sample. By “coated” is meant that a layer ofmicrobe-binding molecules is present on a surface of the solid substrateand available for binding with a microbe. A solid substrate or a solidsupporting structure coated with microbe-binding molecules is alsoreferred to as a “microbe-binding substrate.” The amount of themicrobe-binding molecules used to coat a solid substrate surface canvary with a number of factors such as a solid substrate surface area,coating density, types of microbe-binding molecules, and bindingperformance. A skilled artisan can determine the optimum density ofmicrobe-binding molecules on a solid substrate surface using any methodsknown in the art. By way of example only, the amount of themicrobe-binding molecules used to coat a solid substrate can vary fromabout 1 wt % to about 30 wt % or from about 5 wt % to about 20 wt %. Insome embodiments, the amount of the microbe-binding molecules used tocoat the solid substrate can be higher or lower, depending on a specificneed. However, it should be noted that if the amount of themicrobe-binding molecules used to coat the substrate is too low, themicrobe-binding substrate can show a lower binding performance with amicrobe. On the contrary, if the amount of the microbe-binding moleculesused to coat the substrate is too high, the dense layer of themicrobe-binding molecules can exert an adverse influence on the bindingproperties.

In some embodiments, the microbe-binding substrate is a particle, e.g.,a nano- or micro-particle. In some embodiments, the microbe-bindingmolecule coated substrate is a MBL, a recombinant MBL, FcMBL orAKT-FcMBL coated bead, microbead or magnetic microbead as described inthe International Application Publication Nos. WO/2011/090954(corresponding U.S. patent application Ser. No. 13/574,191 entitled“Engineered opsonin for pathogen detection and treatment”) andWO/2013/012924 (corresponding U.S. patent application Ser. No.14/233,553 entitled “Engineered microbe-targeting molecules and usesthereof”), contents of both of which are incorporated herein byreference. In some embodiments, the microbe-binding substrate can becoated with antibodies, aptamers, or nucleic acids against specificmicrobes, lectin (e.g., but not limited to MBL), or any combinationsthereof.

After addition of the microbe-binding substrate, the microbe-bindingsubstrate can be mixed in the sample to allow microbes to bind with theaffinity molecule. This can be simply accomplished by agitating thesample, e.g., shaking the sample and/or moving the sample around in amicrofluidic device.

In some embodiments, the microbe-binding substrate can be pretreatedwith the blocking agent as described herein, prior to contacting a testsample with the microbe-binding substrate. In some embodiments, ablocking agent and a test sample can be added to the microbe-bindingsubstrate concurrently. The blocking agent is selected to have aneffective binding affinity for the microbe-binding substrate that islower than the effective binding affinity of the microbes and/orfragments thereof for the microbe-binding substrate; and wherein theblocking agent is selected to have the effective binding affinity forthe microbe-binding substrate that is higher than the effective bindingaffinity of at least one interfering agent present in the sample for themicrobe-binding substrate. In these embodiments, the microbes orfragments thereof displaces the blocking agent bound to the coatedsubstrates (microbe-binding molecules) and becomes captured on thecoated substrates.

The high affinity binding of the captured microorganism can displace theblocking agent (e.g., sugar such as glucose) and prevent undesirablebinding of blood cells and/or other non-target materials that wouldinterfere with downstream detection/analysis processes, e.g., ATP-baseddetection of viable bacteria or downstream genetic amplificationefficiency or immunoenzymatic detection. While performing an assay usinga dialysis-like therapeutic (DLT) device, e.g., as described in theInternational Application Publication No. WO 2012/135834, the content ofwhich is incorporated herein by reference, FcMBL beads or membrane canbe preloaded with a blocking agent (e.g., but not limited to, glucose ormaltose) to not only enhance the capture of pathogens and microbialcarbohydrate compounds but also to enhance magnetic bead recovery andprevent FcMBL inactivation by low affinity binders such as erythrocytes.

To prevent or reduce agglutination during separation of the microbesfrom the sample, additional reagents can be added to the sample mixture.For example, a reagent can be added to reduce agglutination by bindingwith an empty ligand binding site on the target-binding molecules.

1210 (Microbe separation from sample): The sample mixture is thensubjected to a microbe separation process. Without wishing to be boundby a theory, in some embodiments, capture and separation of the boundmicrobes and/or fragments thereof from the sample can concentrate themicrobes and/or fragments thereof. In some embodiments, capture andseparation of the bound microbes and/or fragments thereof from thesample can deplete microbes and/or fragments thereof from a sample. Insome embodiments, capture and separation of the bound microbes and/orfragments thereof from the sample can remove components which caninterfere with the assay from the bound microbes and/or fragmentsthereof. Any method known in the art for separating the microbe-bindingsubstrate from the sample can be employed.

For example, when the microbe-binding substrate is magnetic, e.g., amagnetic bead, a magnet can be employed to separate the substrate boundmicrobes from the sample fluid. Without limitations, microbe capturealso can be carried out by non-magnetic means, for example, by coatingmicrobe-binding molecules on non-magnetic solid substrates or scaffolds(e.g., beads, posts, fibers, filters, capillary tubes, etc.) and flowsample by these affinity substrates.

The skilled artisan is well aware of methods for carrying out magneticseparations. Generally, a magnetic field or magnetic field gradient canbe applied to direct the magnetic beads. Optionally, the bound microbecan be washed with a buffer to remove any leftover sample and unboundcomponents. Without wishing to be bound by a theory, capture andseparation of the bound microbes from the sample can concentrate themicrobes and also remove components, which can interfere with the assayor process, from the test sample.

In some embodiments where the microbe-binding agent is in a form ofmagnetic particles, a mixture of smaller microbe-binding magneticparticles and larger magnetic particles can be added to a test sample.The larger magnetic particles can act as local magnetic field gradientconcentrators, thereby attracting the smaller microbe-boundmicrobe-binding magnetic particles to the larger magnetic particles andforming an aggregate, which in turn can be immobilized in the presenceof a magnetic field gradient more readily than individual smallermicrobe-binding magnetic particles. Thus, addition of magnetic particlesthat are larger than the microbe-binding magnetic particles can reduceloss of smaller microbe-bound microbe-binding particles to a fluidduring each wash and/or magnetic separation. This concept of usinglarger magnetic particles to act as local magnetic field gradientconcentrators can be extended to magnetic separation of anytarget-binding magnetic particles, and is described in U.S. ProvisionalAppl. No. 61/772,436 entitled “Methods for Magnetic Capture of a TargetMolecule,” the content of which is incorporated herein by reference.

In some embodiments, the magnetic field gradient can be generated by amagnetic field gradient generator described in the U.S. ProvisionalApplication No. 61/772,360, entitled “Magnetic Separator.”

In some embodiments, microbe capture and/or separation can be performedby flowing a test sample through a device comprising (i) a chamber withan inlet and an outlet, (ii) at least one capture element disposed inthe chamber between the inlet and outlet, wherein the capture elementhas on its surface at least one microbe-binding molecule (e.g., FcMBL).An exemplary capture element can include, but is not limited to, amixing element (e.g., a static mixer or a spiral mixer). Examples ofsuch devices and methods of use are described in U.S. Provisional No.61/673,071, entitled “Devices for Capturing a Microbe or MicrobialMatter,” the content of which is incorporated herein by reference.

In some embodiments, microbe capture and/or microbe-binding substrateseparation can be performed by a rapid microbe diagnostic device asdescribed in Int. Pat. App. No. WO 2011/091037, filed Jan. 19, 2011(corresponding U.S. application Ser. No. 13/522,800), and/or WO2012/135834 filed Apr. 02, 2012 (corresponding U.S. application Ser. No.14/007,738), the contents of all of which are incorporated herein byreference. A rapid microbe diagnostic device as described in Int. Pat.App. No. WO 2011/091037, filed Jan. 19, 2011 (corresponding U.S.application Ser. No. 13/522,800), the content of which is incorporatedherein by reference, can be modified to replace the capture chamber orcapture and visualization chamber with an s-shaped flow path. A magnetcan then be used to capture bound microbe against the flow path wall;separating the bound microbe from rest of the sample.

Methods of separating or concentrating a microbe (e.g., a pathogen) froma biological sample are also described in the International ApplicationPublication No. WO/2013/012924, (corresponding U.S. patent applicationSer. No. 14/233,553), contents of which are incorporated herein byreference.

Without limitations, if a microbe-binding substrate does not possess amagnetic property, isolation of a microbe-binding substrate (e.g.,particles, posts, fibers, dipsticks, membrane, filters, capillary tubes,etc.) from the test sample can be carried out by non-magnetic means,e.g., centrifugation, and filtration. In some embodiments where themicrobe-binding substrate is in a form a dipstick or membrane, themicrobe-binding dipstick or membrane can be simply removed from the testsample, where microbes, if any, in the test sample, remained bound tothe engineered microbe-binding molecules conjugated to the dipstick ormembrane substrate.

The extracted sample can optionally be washed any number (e.g., 1, 2, 3,4, 5 or more) of times before microbial detection and/or incubation withan antibiotic agent, if desired, for antibiotic susceptibility testing.Without wishing to be bound by a theory, such washing can reduce and oreliminate any contaminants from the biological fluid that can beproblematic during incubation or detection. In one embodiment, themicrobe-binding substrate after isolated from the solution and/or thetest sample can be washed with a buffer (e.g., but not limited to, TBST)for at least about 1-3 times.

Any art-recognized wash buffer that does not affect function/viabilityof the microbe bound on the microbe-binding substrate and does notinterfere with binding of the microbe with the microbe-binding substratecan be used to wash the extracted or isolated microbe-boundmicrobe-binding substrates (e.g., but not limited to microbe-boundmicrobe-binding magnetic particles). Examples of a wash buffer caninclude, but are not limited to, phosphate-buffered saline,Tris-buffered saline (TBS), and a combination thereof. In someembodiments, the same processing buffer described herein withoutmicrobe-binding substrates (e.g., microbe-binding magnetic particles)and microbes can be used as the wash buffer. For example, in someembodiments, a wash buffer can include a mixture of TBS, 0.1%polysorbate and 5 mM Ca2+.

The amount of calcium ions (Ca2+) present in the processing bufferand/or wash buffer can vary from about 1 mM to about 100 mM, from about3 mM to about 50 mM, or from about 5 mM to about 25 mM. Calcium ions canbe obtained from any calcium salts, e.g., but not limited to, CaCl₂,CaBr₂, CaI₂, and Ca(NO₃)₂, and any other art-recognized calcium salts.Without wishing to be bound by theory, the presence of calcium ions inthe processing buffer and/or wash buffer can facilitate and/or maintaincalcium-dependent binding (e.g., lectin-mediated binding such asMBL-mediated binding) of the microbe to a microbe-binding substrate.

In some embodiments, the processing buffer and/or wash buffer canexclude calcium ions and/or include a chelator, e.g., but not limitedto, EDTA. In such embodiments, microbes that solely depend oncalcium-dependent binding (e.g., lectin-mediated binding such asMBL-mediated binding) to the microbe-binding substrate will less likelybind to the microbe-binding substrate in the absence of calcium ions.However, microbes (e.g., pathogens such as S. aureus) that at leastpartly depend on non-calcium-dependent interaction (e.g., but notlimited to, protein A/Fc-mediated binding) with the microbe-bindingsubstrate (e.g., FcMBL-coated magnetic particles) can bind to themicrobe-binding substrate in the absence of calcium ions, and additionalinformation can be found, e.g., in the International ApplicationPublication No. WO/2013/012924, or in the U.S. Provisional App. No.61/605,052 filed Feb. 29, 2012, the content of which is incorporatedherein by reference.

In some embodiments, the capture or extraction from the biological fluidor other test samples can be accomplished by a method that does notrequire the identity of the microbe to be known for capture orextraction. This can be accomplished using a solid substrate coated witha broad-spectrum microbe-binding molecule for microbe extraction fromthe test sample. For example, in their previous work, the inventorsdescribed a method for the extraction and concentration of microbes(e.g., pathogens) from blood that does not require prior identificationof pathogen. See PCT Application No. PCT/US2011/021603, filed Jan. 19,2011, content of which is incorporated herein by reference. The methodis based on beads that are coated with mannose binding lectin (MBL) or agenetically engineered version of MBL (FcMBL or Akt-FcMBL). MBL is a keycomponent of the innate immune system, which binds to carbohydratestructures containing mannose, N-acetyl glucosamine and fucose on thesurface of microbes or pathogens and that are not found on mammaliancells. MBL binds to at least 36 species of bacteria (e.g. Gram positive:Staphylococci, MRSA, VRSA, Streptococci, Clostridium; Gram negative:Pseudomonas, E. coli, Klebsiella,), 17 viruses (e.g. CMV, HIV, Ebola,HSV, HepB), 20 fungi (e.g., Candida, Aspergillus, Cryptococcus), and 9parasites (e.g. Malaria, Schistosoma), in addition to at least onemolecular toxin (e.g., LPS endotoxin). Consequently, MBL can serve as abroad-spectrum capture reagent, allowing a wide range of microbes (e.g.,pathogens) to be extracted and concentrated from blood samples or otherbiological fluids.

Accordingly, in some embodiments of the aspects described herein,microbe capture or extraction from a biological sample or other testsample is by substrate coated with a broad-spectrum microbe-bindingmolecule. For example, microbe capture or extraction from a biologicalsample is by magnetic micro- or nano-beads as described in theInternational Application Publication Nos. WO/2011/090954 (correspondingU.S. patent application Ser. No. 13/574,191 entitled “Engineered opsoninfor pathogen detection and treatment”) and WO/2013/012924 (correspondingU.S. patent application Ser. No. 14/233,553 entitled “Engineeredmicrobe-targeting molecules and uses thereof”), contents of both ofwhich are incorporated herein by reference.

In some embodiments, adding a solid substrate coated with ananticoagulant to the extracted microbe sample can allow for bettersample division, analysis or reproducibility. Without wishing to bebound by theory, addition of additional anticoagulant can reduceclumping of microbe-binding substrates. Accordingly, in someembodiments, anticoagulant coated substrate can be added to the testsample before or during or after the capture step. Without limitations,anticoagulant can be coated on a microbe-binding substrate (i.e. a solidsubstrate coated with a microbe-binding molecule). Generally, coatingthe substrate with an anticoagulant before coating with microbe-bindingmolecule provides substantially same efficiency as for a microbe-bindingsubstrate that has not been coated with an anticoagulant. Alternatively,or in addition, a solid substrate coated only with anticoagulant can beadded.

Any amount of anticoagulant coated substrate can be added to the testsample. For example, amount of anticoagulant coated substrate can befrom about 5 wt % to about 500 wt % of the microbe-binding moleculecoated substrate to be used for microbe extraction.

In some embodiments, about equal amounts of anticoagulant coated andmicrobe-binding molecule coated substrate can be added to the testsample.

1212 (Microbe detection/analysis): A detection component, device orsystem can be used to detect and/or analyze the presence of theseparated microbe, for example, by spectroscopy, electrochemicaldetection, polynucleotide detection, fluorescence anisotropy,fluorescence resonance energy transfer, electron transfer, enzyme assay,magnetism, electrical conductivity, isoelectric focusing,chromatography, immunoprecipitation, immunoseparation, aptamer binding,filtration, electrophoresis, use of a CCD camera, immunoassay, ELISA,Gram staining, immunostaining, microscopy, immunofluorescence, westernblot, polymerase chain reaction (PCR), RT-PCR, fluorescence in situhybridization, sequencing, mass spectroscopy, or substantially anycombination thereof. The separated microbe can remain bound on themicrobe-binding substrate during detection and/or analysis, or beisolated form the microbe-binding substrate prior to detection and/oranalysis.

In some embodiments, labeling molecules that can bind with the microbecan also be used to label the microbes for detection. As used herein, a“labeling molecule” refers to a molecule that comprises a detectablelabel and can bind with a target microbe. Labeling molecules caninclude, but are not limited to, MBL or a portion thereof, FcMBL,AKT-FcMBL, wheat germ agglutinin, lectins, antibodies (e.g.,gram-negative antibodies or gram-positive antibodies, antibiotics tospecific microbial strains or species), antigen binding fragments ofantibodies, aptamers, ligands (agonists or antagonists) of cell-surfacereceptors and the like. The labeling molecule can also be a non-specificlabeling molecule that non-specifically stains all viable cells in asample.

As used herein, the term “detectable label” refers to a compositioncapable of producing a detectable signal indicative of the presence of atarget. Detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable labels include fluorescentmolecules, radioisotopes, nucleotide chromophores, enzymes, substrates,chemiluminescent moieties, bioluminescent moieties, and the like. Assuch, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means needed for the methods and devices described herein.

A wide variety of fluorescent reporter dyes are known in the art.Typically, the fluorophore is an aromatic or heteroaromatic compound andcan be a pyrene, anthracene, naphthalene, acridine, stilbene, indole,benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine,salicylate, anthranilate, coumarin, fluorescein, rhodamine or other likecompound.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label.

In some embodiments, the detectable label is a fluorophore or a quantumdot. Without wishing to be bound by a theory, using a fluorescentreagent can reduce signal-to-noise in the imaging/readout, thusmaintaining sensitivity. Accordingly, in some embodiments, prior todetection, the microbes isolated from or remained bound on themicrobe-binding substrate can be stained with at least one stain, e.g.,at least one fluorescent staining reagent comprising a microbe-bindingmolecule, wherein the microbe-binding molecule comprises a fluorophoreor a quantum dot. Examples of fluorescent stains include, but are notlimited to, any microbe-binding element (e.g., microbe-specificantibodies or any microbe-binding proteins or peptides oroligonucleotides) typically conjugated with a fluorophore or quantumdot, and any fluorescent stains used for detection as described herein.

In some embodiments, a labeling molecule can be configured to include a“smart label”, which is undetectable when conjugated to themicrobe-binding molecules, but produces a color change when releasedfrom the engineered molecules in the presence of a microbe enzyme. Thus,when a microbe binds to the engineered microbe-binding molecules, themicrobe releases enzymes that release the detectable label from theengineered molecules. An observation of a color change indicatespresence of the microbe in the sample.

In some embodiments, the microbe-binding substrate can be conjugatedwith a label, such as a detectable label or a biotin label.

In some embodiments, the labeling molecule can comprise MBL or amicrobe-binding molecule described herein. In one embodiment, thelabeling molecule comprises FcMBL. Without wishing to be bound by atheory, labeling molecules based on MBL, and FcMBL in particular, attachselectively to a broad range of microbes, and so they enable the methoddescribed herein to detect the majority of blood-borne microbes withhigh sensitivity and specificity.

Any method known in the art for detecting the particular label can beused for detection. Exemplary methods include, but are not limited to,spectrometry, fluorometry, microscopy imaging, immunoassay, and thelike. While the microbe capture step can specifically capture microbes,it can be beneficial to use a labeling molecule that can enhance thisspecificity. If imaging, e.g., microscopic imaging, is to be used fordetecting the label, the staining can be done either prior to or afterthe microbes have been laid out for microscopic imaging. Additionally,imaging analysis can be performed via automated image acquisition andanalysis.

For optical detection, including fluorescent detection, more than onestain or dye can be used to enhance the detection or identification ofthe microbe. For example, a first dye or stain can be used that can bindwith a genus of microbes, and a second dye or strain can be used thatcan bind with a specific microbe. Colocalization of the two dyes thenprovides enhanced detection or identification of the microbe by reducingfalse positive detection of microbes.

In some embodiments, microscopic imaging can be used to detect signalsfrom label on the labeling agent. Generally, the microbes in thesubsample are stained with a staining reagent and one or more imagestaken from which an artisan can easily count the number of cells presentin a field of view.

In particular embodiments, microbe can be detected through use of one ormore enzyme assays, e.g., enzyme-linked assay (ELISA). Numerous enzymeassays can be used to provide for detection. Examples of such enzymeassays include, but are not limited to, beta-galactosidase assays,peroxidase assays, catalase assays, alkaline phosphatase assays, and thelike. In some embodiments, enzyme assays can be configured such that anenzyme will catalyze a reaction involving an enzyme substrate thatproduces a fluorescent product. Enzymes and fluorescent enzymesubstrates are known and are commercially available (e.g.,Sigma-Aldrich, St. Louis, Mo.). In some embodiments, enzyme assays canbe configured as binding assays that provide for detection of microbe.For example, in some embodiments, a labeling molecule can be conjugatedwith an enzyme for use in the enzyme assay. An enzyme substrate can thenbe introduced to the one or more immobilized enzymes such that theenzymes are able to catalyze a reaction involving the enzyme substrateto produce a detectable signal.

In some embodiments, an enzyme-linked assay (ELISA) can be used todetect signals from the labeling molecule. In ELISA, the labelingmolecule can comprise an enzyme as the detectable label. Each labelingmolecule can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) enzymes. Additionally, each labeling molecule can comprise oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) sites for bindingwith a microbe. Without wishing to be bound by a theory, presence ofmultimeric probe molecules can enhance ELISA signal.

For ELISA, any labeling molecule conjugated to an enzyme can be used.Exemplary labeling molecule include those comprising MBL, FcMBL,AKT-FcMBL, wheat germ agglutinin, lectins, antibodies (e.g.,gram-negative antibodies or gram-positive antibodies), antigen bindingfragments of antibodies, aptamers, ligands (agonists or antagonists) ofcell-surface receptors and the like.

In some embodiments, the labeling molecule can comprise MBL or FcMBLlabeled with a detectable label.

Similarly, a variety of enzymes can be used, with either colorimetric orfluorogenic substrates. In some embodiments, the reporter-enzymeproduces a calorimetric change which can be measured as light absorptionat a particular wavelength. Exemplary enzymes include, but are notlimited to, beta-galactosidases, peroxidases, catalases, alkalinephosphatases, and the like.

In some embodiments, the enzyme is a horseradish peroxidase (HRP).

In some embodiments, the enzyme is an alkaline peroxidase (AP).

A microbe-binding molecule and the enzyme can be linked to each other bya linker. In some embodiments, the linker between the microbe-bindingmolecule and the enzyme is an amide bond. In some embodiments, thelinker between the microbe-binding molecule and the enzyme is adisulfide (S—S) bond.

When the microbe-binding molecule is a peptide, polypeptide or aprotein, the enzyme can be linked at the N-terminus, the C-terminus, orat an internal position of the microbe-binding molecule. Similarly, theenzyme can be linked by its N-terminus, C-terminus, or an internalposition.

In one embodiment, the ELISA probe molecule can comprise a MBL or aportion there of or a FcMBL molecule linked to a HRP. Conjugation of HRPto any proteins and antibodies are known in the art. In one embodiment,FcMBL-HRP construct is generated by direct coupling HRP to FcMBL usingany commercially-available HRP conjugation kit. In some embodiments, themicrobes isolated from or remained bound on the microbe-bindingsubstrate can be incubated with the HRP-labeled microbe-bindingmolecules, e.g., MBL or a portion thereof, or a FcMBL molecule linked toa HRP for a period of time, e.g., at least about 5 mins, at least about10 mins, at least about 15 mins, at least about 20 mins, at least about25 mins, at least about 30 mins. The typical concentrations ofHRP-labeled molecules used in the ELISA assay can range from about 1:500to about 1:20,000 dilutions. In one embodiment, the concentration ofHRP-labeled microbe-binding molecules, e.g., MBL or a portion thereof,or a FcMBL molecule linked to a HRP molecule, can be about 1:1000 toabout 1:10000 dilutions.

In one embodiment, the ELISA probe molecule can comprise a MBL or aportion thereof, or a FcMBL molecule linked to a AP. Conjugation of APto any proteins and antibodies are known in the art. In one embodiment,FcMBL-AP construct is generated by direct coupling AP to FcMBL using anycommercially-available AP conjugation kit. In some embodiments, themicrobes isolated from or remained bound on the microbe-bindingsubstrate can be incubated with the AP-labeled microbe-binding molecule,e.g., MBL or a portion thereof, or a FcMBL molecule linked to a AP for aperiod of time, e.g., at least about 5 mins, at least about 10 mins, atleast about 15 mins, at least about 20 mins, at least about 25 mins, atleast about 30 mins. The typical concentrations of AP-labeled moleculesused in the ELISA assay can range from about 1: 1000 to about 1:20,000dilutions. In one embodiment, the concentration of AP-labeledmicrobe-binding molecules, e.g., MBL or a portion thereof, or a FcMBLmolecule linked to a AP molecule, can be about 1:5000 to about 1:10000dilutions.

Following incubation with the ELISA probe molecules, the sample can bewashed with a wash buffer one or more (e.g., 1, 2, 3, 4, 5 or more)times to remove any unbound probes. An appropriate substrate for theenzyme (e.g., HRP or AP) can be added to develop the assay. Chromogenicsubstrates for the enzymes (e.g., HRP or AP) are known to one of skillin the art. A skilled artisan can select appropriate chromogenicsubstrates for the enzyme, e.g., TMB substrate for the HRP enzyme, orBCIP/NBT for the AP enzyme. In some embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain calcium ionsat a concentration of about at least about 0.01 mM, at least about 0.05mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM,at least about 2.5 mM, at least about 5 mM, at least about 10 mM, atleast about 20 mM, at least about 30 mM, at least about 40 mM, at leastabout 50 mM or more. In alternative embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain no calciumions. In some embodiments, the wash buffer used after incubation with anELISA probe molecule can contain a chelating agent. A wash buffer can beany art-recognized buffer used for washing between incubations withantibodies and/or labeling molecules. An exemplary wash buffer caninclude, but is not limited to, TBST.

In some embodiments, without wishing to be bound by theory, it can bedesirable to use a wash buffer without a surfactant or a detergent forthe last wash before addition of a chromogenic substrate, because asurfactant or detergent may have adverse effect to the enzymaticreaction with a chromogenic substrate.

One advantage of the ELISA-based approach is that the solid substratedoes not need to be dispersed or dissociated from the microbe beforebinding the secondary reagents. This is in contrast to microscopictechniques, in which excess residual solid substrate may obscure themicrobe during imaging. Furthermore, the optical readout components forELISA are likely cheaper than in the microscopy case, and there is noneed for focusing or for demanding that the sample be on the same focalplane. A further advantage of the ELISA-based approach is that it cantake advantage of commercially available laboratory equipment. Inparticular, when the solid substrate is magnetic, magnetic separationcan be automated using the KINGFISHER® system, the brief culture can beperformed using an airlift fermenter, and the colorimetric/fluorescentreadout can be attained using a standard plate reader.

Further amplification of the ELISA signal can be obtained bymultimerizing the recognition molecule (e.g., the microbe-bindingmolecule) or by multimerizing the detection enzyme (HRP, etc.). Forinstance, phage expression can be used to yield multimerized MBL andprovide a scaffold to increase the concentration of HRP (either throughdirect coupling of HRP to the phage particles or using an HRP-antiM13conjugated antibody).

In some embodiments, microbe can be detected through use of immunoassay.Numerous types of detection methods may be used in combination withimmunoassay based methods.

Without limitations, detection of microbes in a sample can also becarried out using light microscopy with phase contrast imaging based onthe characteristic size (5 um diameter), shape (spherical to elliptical)and refractile characteristics of target components such as microbesthat are distinct from all normal blood cells. Greater specificity canbe obtained using optical imaging with fluorescent or cytochemicalstains that are specific for all microbes or specific subclasses (e.g.calcofluor (1 μM to 100 μM) for chitin in fungi, fluorescent antibodiesdirected against fungal surface molecules, gram stains, acid-faststains, fluorescent MBL, fluorescent Fc-MBL, etc.).

In some embodiments, a microbe can be detected through use ofspectroscopy. Numerous types of spectroscopic methods can be used.Examples of such methods include, but are not limited to, ultravioletspectroscopy, visible light spectroscopy, infrared spectroscopy, x-rayspectroscopy, fluorescence spectroscopy, mass spectroscopy, plasmonresonance (e.g., Cherif et al., Clinical Chemistry, 52:255-262 (2006)and U.S. Pat. No. 7,030,989; herein incorporated by reference), nuclearmagnetic resonance spectroscopy, Raman spectroscopy, fluorescencequenching, fluorescence resonance energy transfer, intrinsicfluorescence, ligand fluorescence, and the like.

In some embodiments, a metabolic assay is used to determine the relativenumber of microbes in a sample compared to a control. As will beapparent to one of ordinary skill in the art any metabolic indicatorthat can be associated with cells can be used, such as but not limitedto, turbidity, fluorescent dyes, and redox indicators such as, but notlimited to, Alamar Blue, MTT, XTT, MTS, and WST. Metabolic indicatorscan be components inherent to the cells or components added to theenvironment of the cells. In some embodiments, changes in or the stateof the metabolic indicator can result in alteration of ability of themedia containing the sample to absorb or reflect particular wavelengthsof radiation.

In some embodiments, microbes isolated from or remained bound onmicrobe-binding substrate can be labeled with nucleic acid barcodes forsubsequent detection and/or multiplexing detection. Nucleic acidbarcoding methods for detection of one or more analytes in a sample arewell known in the art.

In other embodiments, the captured microbe can be analyzed and/ordetected in the capture chamber or capture and visualization chamber ofa rapid microbe diagnostic device described in the Int. Pat. App. No. WO2011/091037, filed Jan. 19, 2011 (corresponding U.S. application Ser.No. 13/522,800), the contents of which are incorporated herein byreference. Alternatively, the captured microbe can be recovered (i.e.,removed) and analyzed and/or detected.

In some embodiments, the captured microbe is recovered and analyzedand/or detected using a particle on membrane assay as described in U.S.Pat. No. 7,781,226, content of which is incorporated herein byreference. A particle on membrane assay as described in U.S. Pat. No.7,781,226 can be operably linked with a rapid microbe diagnostic deviceof the Int. Pat. App. No. WO 2011/091037 (corresponding U.S. applicationSer. No. 13/522,800), the contents of which are incorporated herein byreference, to reduce the number of sample handling steps, automate theprocess and/or integrate the capture, separation and analysis/detectionsteps into a microfluidic device.

In some embodiments, microbe capture, separation and analysis can bedone using a hybrid microfluidic SPR and molecular imagining device asdescribed in U.S. Pat. App. Pub. No. 2011/0039280.

In some embodiments, microbe detection and/or identification can use oneor more embodiments of the compositions and/or methods described in theInternational Application No. PCT/US12/71398 filed Dec. 21, 2012,content of which is incorporated herein by reference.

In some embodiments, the processes or assays described herein can detectthe presence or absence of a microbe and/or identify a microbe in a testsample in less than 24 hours, less than 12 hours, less than 10 hours,less than 8 hours, less than 6 hours, less than 4 hours, less than 3hours, less than 2 hours, less than 1 hour, or lower. In someembodiments, the processes or assays described herein can detect thepresence or absence of a microbe and/or identify a microbe in a testsample in less than 6 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 1 hour, or lower.

Optional additional analyses or treatment—culturing: In some embodimentsof any aspects described herein, the assay or process can furthercomprise culturing any microbe bound on the microbe-binding substrate(e.g., microbe-binding magnetic microbeads) for a period of time. Insuch embodiments, the microbe bound on the microbe-binding substrate canexpand in population by at least about 10% after culturing for a periodof time.

In some embodiments, the microbes bound on the microbe-bindingsubstrates (e.g., microbe-binding magnetic microbeads) can be culturedon a microbe-compatible culture medium, e.g., plated on an agar plate orcultured in LB broth. One of skill in the art will readily recognizemicrobial culture techniques, including, but not limited to, the use ofincubators and/or equipment used to provide a gentle agitation, e.g.,rotator platforms, and shakers, if necessary, e.g., to prevent the cellsfrom aggregation without subjecting them to a significant shear stressand provide aerial agitation.

The microbes can remain bound on the microbe-binding substrate (e.g.,microbe-binding magnetic microbeads) during detection and/or additionalanalyses described herein or they can be detached, eluted off or removedfrom a microbe-binding substrate prior to detection or additionalanalyses described herein. In some embodiments where the bound microbesare desired to be detached, eluted off or removed from a microbe-bindingsubstrate, the microbe-binding molecules of the microbe-bindingsubstrate can be further contacted with a low pH buffer, e.g., a pHbuffer less than 6, less than 5, less than 4, less than 3, less than 2,less than 1 or lower. In some embodiments, a low pH buffer that does notcause precipitation of a chelating agent, if present, can be used. Inone embodiment, a low pH buffer can be arginine. In another embodiment,a low pH buffer can be pyrophosphate.

In some embodiments of any aspects described herein, the microbe-bindingmolecules of the microbe-binding substrate can be further contacted witha low pH buffer and a chelating agent. In some embodiments, the contactof the microbe-binding molecules of the microbe-binding substrate withthe low pH buffer and the chelating agent can be concurrent orsequentially. In one embodiment, the microbe-binding molecules of themicrobe-binding substrate can be further contacted with arginine (e.g.,2 M) with EDTA or EGTA at pH 4.4.

The isolated microbes can then be used for analyses described earlier oradditional treatment, e.g., expansion in culture, antibiotic sensitivitytesting, sequencing and/or DNA or RNA analysis.

Optional additional analyses or treatment—antibiotic sensitivity orsusceptibility testing: In some embodiments of any aspects describedherein, the process or assay described herein can further comprisesubjecting the microbes bound on the microbe-binding substrate (e.g.,microbe-binding magnetic microbeads) and/or the expanded cultures ofmicrobes isolated from the microbe-binding substrate (e.g.,microbe-binding magnetic microbeads) to one or more antibiotics. Theresponse of the microbe to an antibiotic can then be evaluated with anyknown methods in the art, e.g., by measuring the viability of microbes.Thus, an appropriate antibiotic can be identified for treatment of aninfection caused by a microbe, even though the specific species of themicrobe bound onto the microbe-binding substrate is initially unknown.Additional details for use of engineered microbe-binding moleculesdescribed herein in antibiotic sensitivity testings can be found, e.g.,in the International Application No. PCT/US13/28409 filed Feb. 28, 2013.

Any processes or steps described herein can be performed by a module ordevice. While these are discussed as discrete processes, one or more ofthe processes or steps described herein can be combined into one systemfor carrying out the assays of any aspects described herein.

In some embodiments, the assay or process 1200 described herein can beadapted for use in a high-throughput platform, e.g., an automated systemor platform.

Microbe-Binding Agents or Molecules

In some embodiments, the target-binding agents comprise microbe-bindingagents or molecules. In some embodiments, the target-binding moleculescomprise microbe-binding molecules. In some embodiments, the blockingagent is pre-bound to the microbe-binding agents or microbe-bindingmolecules. Any molecule or material that can bind to a microbe can beemployed as the microbe-binding molecule. Exemplary microbe-bindingmolecules (or microbe-binding molecules) include, but are not limitedto, opsonins, lectins, antibodies and antigen binding fragments thereof,proteins, peptides, nucleic acids, carbohydrates, lipids, and anycombinations thereof. The microbe-binding molecule can comprise at leastone (e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more) microbe surface-binding domain(“microbe binding domain”). The term “microbe surface-binding domain” asused herein refers to any molecules or a fragment thereof that canspecifically bind to the surface of a microbe, e.g., any componentpresent on a surface of a microbe.

Materials or substances which can serve as microbe-binding moleculesinclude, for example, peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, smallmolecules, and any combinations thereof. The microbe-binding moleculecan be covalently (e.g., cross-linked) or non-covalently linked to thesubstrate surface.

In some embodiments, the microbe surface-binding domain can comprise anopsonin or a fragment thereof. The term “opsonin” as used herein refersto naturally-occurring and synthetic molecules which are capable ofbinding to or attaching to the surface of a microbe or a pathogen, ofacting as binding enhancers for a process of phagocytosis. Examples ofopsonins which can be used in the engineered molecules described hereininclude, but are not limited to, vitronectin, fibronectin, complementcomponents such as Clq (including any of its component polypeptidechains A, B and C), complement fragments such as C3d, C3b and C4b,mannose-binding protein, conglutinin, surfactant proteins A and D,C-reactive protein (CRP), alpha2-macroglobulin, and immunoglobulins, forexample, the Fc portion of an immunoglobulin.

In some embodiments, the microbe surface-binding domain comprises acarbohydrate recognition domain or a carbohydrate recognition portionthereof. As used herein, the term “carbohydrate recognition domain”refers to a region, at least a portion of which, can bind tocarbohydrates on a surface of a microbe (e.g., a pathogen).

In some embodiments, the microbe surface-binding domain comprises alectin or a carbohydrate recognition or binding fragment or portionthereof. The term “lectin” as used herein refers to any moleculesincluding proteins, natural or genetically modified, that interactspecifically with saccharides (i.e., carbohydrates). The term “lectin”as used herein can also refer to lectins derived from any species,including, but not limited to, plants, animals, insects andmicroorganisms, having a desired carbohydrate binding specificity.Examples of plant lectins include, but are not limited to, theLeguminosae lectin family, such as ConA, soybean agglutinin, peanutlectin, lentil lectin, and Galanthus nivalis agglutinin (GNA) from theGalanthus (snowdrop) plant. Other examples of plant lectins are theGramineae and Solanaceae families of lectins. Examples of animal lectinsinclude, but are not limited to, any known lectin of the major groupsS-type lectins, C-type lectins, P-type lectins, and I-type lectins, andgalectins. In some embodiments, the carbohydrate recognition domain canbe derived from a C-type lectin, or a fragment thereof. C-type lectincan include any carbohydrate-binding protein that requires calcium forbinding. In some embodiments, the C-type lectin can include, but are notlimited to, collectin, DC-SIGN, and fragments thereof. Without wishingto be bound by theory, DC-SIGN can generally bind various microbes byrecognizing high-mannose-containing glycoproteins on their envelopesand/or function as a receptor for several viruses such as HIV andHepatitis C.

In some embodiments, the microbe-binding molecules or microbe-bindingmolecules can comprise a microbe-binding portion of the C-type lectins,including, e.g., but not limited to, soluble factors such as Collectins(e.g., MBL, surfactant protein A, surfactant protein D and Collectin11), ficolins (e.g. L-Ficolin, Ficolin A), receptor based lectins (e.g.,DC-SIGN, DC-SIGNR, SIGNR1, Macrophage Mannose Receptor 1, Dectin-1 andDectin-2), lectins from the shrimp Marsupenaeus japonicus (e.g. LectinA, Lectin B and Lectin C), or any combinations thereof.

In some embodiments, the microbe-binding molecules can comprise at leasta portion of non-C-type lectins (e.g., but not limited to, Wheat GermAgglutinin).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of lipopolysaccharide (LPS)-binding proteins and/or endotoxinbinding proteins (e.g., but not limited to, CD14, MD2,lipopolysaccharide binding proteins (LBP), limulus anti-LPS factor(LAL-F), or any combinations thereof).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of peptidoglycan binding proteins (e.g., but not limited to,mammalian peptidoglycan recognition protein-1 (PGRP-1), PGRP-2, PGRP-3,PGRP-4, or any combinations thereof.

Collectins are soluble pattern recognition receptors (PRRs) belonging tothe superfamily of collagen containing C-type lectins. Exemplarycollectins include, without limitations, mannan-binding lectin (MBL) ormannose-binding protein, surfactant protein A (SP-A), surfactant proteinD (SP-D), collectin liver 1 (CL-L1), collectin placenta 1 (CL-P1),conglutinin, collectin of 43 kDa (CL-43), collectin of 46 kDa (CL-46),and a fragment thereof.

In some embodiments, the microbe-surface binding domain comprises thefull amino acid sequence of a carbohydrate-binding protein. In someembodiments, the microbe-surface binding domain comprises a sequence ofa carbohydrate recognition domain of a carbohydrate-binding protein.Examples of carbohydrate-binding proteins include, but are not limitedto, lectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, glucose-bindingprotein, Galanthus nivalis agglutinin, peanut lectin, lentil lectin,DC-SIGN, C-reactive protein (CRP), and any combinations thereof.

In some embodiments, the microbe surface-binding molecule comprises amannose-binding lectin (MBL) or a carbohydrate binding fragment orportion thereof. Mannose-binding lectin, also called mannose bindingprotein (MBP), is a calcium-dependent serum protein that can play a rolein the innate immune response by binding to carbohydrates on the surfaceof a wide range of microbes or pathogens (viruses, bacteria, fungi,protozoa) where it can activate the complement system. MBL can alsoserve as a direct opsonin and mediate binding and uptake of microbes orpathogens by tagging the surface of a microbe or pathogen to facilitaterecognition and ingestion by phagocytes. MBL and an engineered form ofMBL (FcMBL and Akt-FcMBL) are described in the International ApplicationPublication Nos. WO/2011/090954 (corresponding U.S. patent applicationSer. No. 13/574,191 entitled “Engineered opsonin for pathogen detectionand treatment”) and WO/2013/012924 (corresponding U.S. patentapplication Ser. No. 14/233,553 entitled “Engineered microbe-targetingmolecules and uses thereof”), contents of both of which are incorporatedherein by reference.

In some embodiments, the microbe surface-binding molecule comprises atleast a portion of C-reactive protein that binds to a microbe orfragment thereof. Microbe-binding molecules comprising a portion ofC-reactive protein described in U.S. Provisional App. No. 61/917,705entitled “CRP Capture/Detection of Gram Positive Bacteria,” the contentsof which are incorporated herein by reference.

Without wishing to be bound by a theory, microbe binding moleculescomprising lectins or modified versions thereof can act asbroad-spectrum microbe binding molecules (e.g., pathogen bindingmolecules). Accordingly, antibiotic susceptibility method utilizinglectins (e.g., MBL and genetically engineered version of MBL (FcMBL andAkt-FcMBL)) as broad-spectrum microbe binding molecules (e.g., pathogenbinding molecules) to capture and grow the microbes, can be carried outwithout identifying the microbe (e.g., pathogen), either for extractionor for antibiotic sensitivity testing.

In some embodiments, at least two microbe surface-binding domains (e.g.two, three, four, five, six, seven or more) microbe surface-bindingdomains, can be linked together to form a multimeric microbesurface-binding domain. In such embodiments, the distances betweenmicrobe surface-binding domains can be engineered to match with thedistance between the binding sites on the target microbe surface. Insome embodiments, the microbe surface-binding domain can be present in aform of a monomer, dimer, trimer, tetramer, pentamer, hexamer, or anentity comprising more than six sub-units.

A multimeric microbe surface-binding domain can have each of theindividual microbe surface-binding domains be identical. Alternatively,a multimeric microbe surface-binding domain can have at least one, atleast two, or at least three microbe surface-binding domains differentfrom the rest. In such embodiments, microbe surface-binding domains thatshare a common binding specificity for molecule on a microbe surface canbe used. By way of example only, the fibrinogen-like domain of severallectins has a similar function to the CRD of C-type lectins includingMBL, and function as pattern-recognition receptors to discriminatemicrobes or pathogens from self. One of such lectins comprising thefibrinogen-like domain is serum ficolins.

Serum ficolins have a common binding specificity for GlcNAc(N-acetyl-glucosamine), elastin or GalNAc (N-acetyl-galactosamine). Thefibrinogen-like domain is responsible for the carbohydrate binding. Inhuman serum, two types of ficolin, known as L-ficolin (also called P35,ficolin L, ficolin 2 or hucolin) and H-ficolin (also called Hakataantigen, ficolin 3 or thermolabile b2-macroglycoprotein), have beenidentified, and both of them have lectin activity. L-ficolin recognizesGlcNAc and H-ficolin recognizes GalNAc. Another ficolin known asM-ficolin (also called P3 5-related protein, ficolin 1 or ficolin A) isnot considered to be a serum protein and is found in leucocytes and inthe lungs. L-ficolin and H-ficolin activate the lectin-complementpathway in association with MASPs. M-Ficolin, L-ficolin and H-ficolinhave calcium-independent lectin activity. Accordingly, in someembodiments, a microbe-binding molecule can comprise MBL and L-ficolincarbohydrate recognition domains, MBL and H-ficolin carbohydraterecognition domains, or a combination thereof.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can also be used in the microbe-bindingmolecules. For example, recombinant mannose-binding lectins, e.g., butnot limited to, the ones disclosed in the U.S. Pat. Nos. 5,270,199;6,846,649; and U.S. Patent App. Publication No. 2004/0229212, contentsof all of which are incorporated herein by reference, can be used inconstructing a microbe-binding molecule.

The microbe binding molecule can further comprise at least one (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty or more) substrate surface binding domain (“substratebinding domain”) adapted for orienting the microbe binding domain awayfrom the substrate surface. As used herein, the term “substrate-bindingdomain” refers to any molecule that facilitates the conjugation of theengineered molecules described herein to a solid substrate or afunctionalized substrate. The microbe binding domain and the substratebinding domains can be linked by a linker. Similarly, the substratebinding domain and the substrate surface can be linked by a linker.

The substrate-binding domain can comprise at least one amino group thatcan non-covalently or covalently couple with functional groups on thesurface of the substrate. For example, the primary amines of the aminoacid residues (e.g., lysine or cysteine residues) at the N-terminus orin close proximity to the N-terminus of the microbe surface-bindingdomains can be used to couple with functional groups on the substratesurface.

In some embodiments, the substrate-binding domain can comprise at leastone, at least two, at least three or more oligopeptides. The length ofthe oligonucleotide can vary from about 2 amino acid residues to about10 amino acid residues, or about 2 amino acid residues to about 5 aminoacid residues. Determination of an appropriate amino acid sequence ofthe oligonucleotide for binding with different substrates is well withinone of skill in the art. For example, an oligopeptide comprising anamino acid sequence of Alanine-Lysine-Threonine (AKT), which provides asingle biotinylation site for subsequent binding to streptavidin-coatedsubstrate. Such single biotinylation site can also enable the microbesurface binding domain of a microbe binding molecule to orient away fromthe substrate, and thus become more accessible to microbes or pathogens.See, for example, Witus et al. (2010) JACS 132: 16812.

In some embodiments, the substrate-binding domain can comprise at leastone oligonucleotide. The sequence and length of the oligonucleotides canbe configured according to the types of the substrate, binding density,and/or desired binding strength. For example, if the substrate is anucleic acid scaffold, e.g., a DNA scaffold, the oligonucleotidesequence of the substrate-binding domain can be designed such that it iscomplementary to a sub-sequence of the nucleic acid scaffold to wherethe substrate-binding domain can hybridize.

In some embodiments, the oligonucleotides can include aptamers. As usedherein, the term “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules by a mechanism otherthan Watson-Crick base pairing or triplex formation. Aptamers caninclude, without limitation, defined sequence segments and sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges. Methods for selecting aptamers for binding to a molecule arewidely known in the art and easily accessible to one of ordinary skillin the art. The oligonucleotides including aptamers can be of anylength, e.g., from about 1 nucleotide to about 100 nucleotides, fromabout 5 nucleotides to about 50 nucleotides, or from about 10nucleotides to about 25 nucleotides. Generally, a longer oligonucleotidefor hybridization to a nucleic acid scaffold can generate a strongerbinding strength between the engineered microbe surface-binding domainand substrate.

The microbe-binding molecules can contain sequences from the samespecies or from different species. For example, an interspecies hybridmicrobe-binding molecule can contain a linker, e.g., a peptide linker,from a murine species, and a human sequence from a carbohydraterecognition domain protein, provided that they do not provideunacceptable levels of deleterious effects. The engineeredmicrobe-binding molecules described herein can also include those thatare made entirely from murine-derived sequences or fully human.

General methods of preparing such microbe-binding molecules are wellknown in the art (Ashkenazi, A. and S. M. Chamow (1997), “Immunoadhesinsas research tools and therapeutic agents,” Curr. Opin. Immunol. 9(2):195-200, Chamow, S. M. and A. Ashkenazi (1996). “Immunoadhesins:principles and applications,” Trends Biotechnol. 14(2):52-60). In oneexample, an engineered microbe-binding molecule can be made by cloninginto an expression vector such as Fc-X vector as discussed in Lo et al.(1998) 11:495 and PCT application no. PCT/US2011/021603, filed Jan. 19,2011, content of both of which is incorporated herein by reference.

In some embodiments, the microbe-binding molecule is a fusion protein orpeptide comprising (a) a carbohydrate recognition domain derived from acarbohydrate binding protein, and (b) a linker as defined below. In someembodiments, the fusion protein or peptide further comprise a substratebinding domain at one of its terminus (e.g., N-terminus), which permitsa microbe-binding molecule to attach to a solid substrate such that thecarbohydrate recognition domain points away from the solid substratesurface.

In one embodiment, the microbe-binding molecule comprises an MBL, acarbohydrate recognition domain of an MBL, or a genetically engineeredversion of MBL (FcMBL) as described in the International ApplicationPublication Nos. WO/2011/090954 (corresponding U.S. patent applicationSer. No. 13/574,191 entitled “Engineered opsonin for pathogen detectionand treatment”) and WO/2013/012924 (corresponding U.S. patentapplication Ser. No. 14/233,553 entitled “Engineered microbe-targetingmolecules and uses thereof”), contents of both of which are incorporatedherein by reference. Amino acid sequences for MBL and engineered MBLare:

(i) MBL full length (SEQ ID NO. 1):MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSSPGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPGNPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMARIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQASVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTGNRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI(ii) MBL without the signal sequence (SEQ ID NO. 2):ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKGEPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKSPDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (iii) Truncated MBL (SEQ ID NO. 3):AASERKALQT EMARIKKWLT FSLGKQVGNK FFLTNGEIMTFEKVKALCVK FQASVATPRN AAENGAIQNL IKEEAFLGITDEKTEGQFVD LTGNRLTYTN WNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP I(iv) Carbohydrate recognition domain (CRD) of MBL (SEQ ID NO. 4):VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGAIQNLIKEEAF LGITDEKTEG QFVDLTGNRL TYTNWNEGEPNNAGSDEDCV LLLKNGQWND VPCSTSHLAV CEFPI (v) Neck +Carbohydrate recognition domain of MBL (SEQ ID NO. 5):PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (vi) FcMBL.81 (SEQ ID NO. 6):EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSPGAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI (vii) Akt-FcMBL (SEQ ID NO. 7):AKTEPKSSDKTHT CPPCPAPELL GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSPGAPDGDSSLA ASERKALQTE MARIKKWLTF SLGKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI (viii) FcMBL.111 (SEQ ID NO. 8):EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GATSKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI

In some embodiments, microbe-binding molecule comprises an amino acidsequence selected from SEQ ID NO. 1-SEQ ID NO. 8.

In some embodiments, microbe-binding agent is a “microbe-bindingsubstrate” as defined herein.

Linkers

As used herein, the term “linker” generally refers to a molecular entitythat can directly or indirectly connect two parts of a composition,e.g., at least one microbe-binding molecule and at least onesubstrate-binding domain or at least one enzyme and at least onemicrobe-binding molecule. In some embodiments, the linker can directlyor indirectly connect to one or more microbe-binding molecule ormicrobe-binding domain.

Linkers can be configures according to a specific need, e.g., based onat least one of the following characteristics. By way of example only,in some embodiments, linkers can be configured to have a sufficientlength and flexibility such that it can allow for a microbesurface-binding domain to orient accordingly with respect to at leastone carbohydrate on a microbe surface. In some embodiments, linkers canbe configured to allow multimerization of at least two engineeredmicrobe-binding molecules (e.g., to from a di-, tri-, tetra-, penta-, orhigher multimeric complex) while retaining biological activity (e.g.,microbe-binding activity). In some embodiments, linkers can beconfigured to facilitate expression and purification of the engineeredmicrobe-binding molecule described herein. In some embodiments, linkerscan be configured to provide at least one recognition-site for proteasesor nucleases. In addition, linkers should be non-reactive with thefunctional components of the engineered molecule described herein (e.g.,minimal hydrophobic or charged character to react with the functionalprotein domains such as a microbe surface-binding domain or asubstrate-binding domain).

In some embodiments, a linker can be configured to have any length in aform of a peptide, a protein, a nucleic acid (e.g., DNA or RNA), or anycombinations thereof. In some embodiments, the peptide or nucleic acidlinker can vary from about 1 to about 1000 amino acids long, from about10 to about 500 amino acids long, from about 30 to about 300 amino acidslong, or from about 50 to about 150 amino acids long. Longer or shorterlinker sequences can be also used for the engineered microbe-bindingmolecules described herein. In one embodiment, the peptide linker has anamino acid sequence of about 200 to 300 amino acids in length.

In some embodiments, a peptide or nucleic acid linker can be configuredto have a sequence comprising at least one of the amino acids selectedfrom the group consisting of glycine (Gly), serine (Ser), asparagine(Asn), threonine (Thr), methionine (Met) or alanine (Ala), or at leastone of codon sequences encoding the aforementioned amino acids (i.e.,Gly, Ser, Asn, Thr, Met or Ala). Such amino acids and correspondingnucleic acid sequences are generally used to provide flexibility of alinker. However, in some embodiments, other uncharged polar amino acids(e.g., Gln, Cys or Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro,Phe, and Trp), or nucleic acid sequences encoding the amino acidsthereof can also be included in a linker sequence. In alternativeembodiments, polar amino acids or nucleic acid sequence thereof can beadded to modulate the flexibility of a linker. One of skill in the artcan control flexibility of a linker by varying the types and numbers ofresidues in the linker. See, e.g., Perham, 30 Biochem. 8501 (1991);Wriggers et al., 80 Biopolymers 736 (2005).

In alternative embodiments, a linker can be a chemical linker of anylength. In some embodiments, chemical linkers can comprise a direct bondor an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH,SO, SO2, SO2NH, or a chain of atoms, such as substituted orunsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl,substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstitutedC6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substitutedor unsubstituted C5-C12 heterocyclyl, substituted or unsubstitutedC3-C12 cycloalkyl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO2, NH, or C(O). In some embodiments, thechemical linker can be a polymer chain (branched or linear).

In some embodiments where the linker is a peptide, such peptide linkercan comprise at least a portion of an immunoglobulin, e.g., IgA, IgD,IgE, IgG and IgM including their subclasses (e.g., IgG1), or a modifiedthereof. In some embodiments, the peptide linker can comprise a portionof fragment crystallization (Fc) region of an immunoglobulin or amodified thereof. In such embodiments, the portion of the Fc region thatcan be used as a linker can comprise at least one region selected fromthe group consisting of a hinge region, a CH2 region, a CH3 region, andany combinations thereof. By way of example, in some embodiments, a CH2region can be excluded from the portion of the Fc region as a linker. Inone embodiment, Fc linker comprises a hinge region, a CH2 domain and aCH3 domain. Such Fc linker can be used to facilitate expression andpurification of the engineered microbe-binding molecules describedherein. The N terminal Fc has been shown to improve expression levels,protein folding and secretion of the fusion partner. In addition, the Fchas a staphylococcal protein A binding site, which can be used forone-step purification protein A affinity chromatography. See Lo KM etal. (1998) Protein Eng. 11: 495-500. Further, such Fc linker have amolecule weight above a renal threshold of about 45 kDa, thus reducingthe possibility of engineered microbe-binding molecules being removed byglomerular filtration. Additionally, the Fc linker can allowdimerization of two engineered microbe-binding molecules to form adimer, e.g., a dimeric MBL molecule.

In various embodiments, the N-terminus or the C-terminus of the linker,e.g., the portion of the Fc region, can be modified. By way of exampleonly, the N-terminus or the C-terminus of the linker can be extended byat least one additional linker described herein, e.g., to providefurther flexibility, or to attach additional molecules. In someembodiments, the N-terminus of the linker can be linked directly orindirectly (via an additional linker) with a substrate-binding domainadapted for orienting the carbohydrate recognition domain away from thesubstrate. Exemplary Fc linked MBL (FcMBL and Akt-FcMBL) are describedin PCT application no. PCT/US2011/021603, filed Jan. 19, 2011, contentof which is incorporated herein by reference.

In some embodiments, the linker can be embodied as part of the microbesurface-binding domain, or part of the microbe surface-binding domain.

In some embodiments, the distance between the microbe surface-bindingdomain and the substrate surface can range from about 50 angstroms toabout 5000 angstroms, from about 100 angstroms to about 2500 angstroms,or from about 200 angstroms to about 1000 angstroms.

In some embodiments, the linkers can be branched. For branched linkers,the linker can linked together at least one (e.g., one, two, three,four, five, six, seven, eight, nine, ten or more) surface binding domainand at least one (e.g., one, two, three, four, five, six, seven, eight,nine, ten or more) microbe surface-binding domain.

In some embodiments provided herein, the linker can further comprise adetectable label. In some embodiments, the detectable label can be achromogenic or fluorogenic microbe enzyme substrate so that when amicrobe binds to the engineered microbe-binding molecule, the enzymethat the microbe releases can interact with the detectable label toinduce a color change. Examples of such microbe enzyme substrate caninclude, but are not limited to, indoxyl butyrate, indoxyl glucoside,esculin, magneta glucoside, red-β-glucuronide,2-methoxy-4-(2-nitrovinyl) phenyl β-D-glu-copyranoside,2-methoxy-4-(2-nitrovinyl) phenyl β-D-cetamindo-2-deoxyglucopyranoside,and any other art-recognized microbe enzyme substrates. Such embodimentscan act as an indicator for the presence of a microbe or pathogen.

Exemplary Microbes or Pathogens that can be Detected or Captured usingthe Methods, Compositions, Kits and Systems Described Herein

As used interchangeably herein, the terms “microbes” and “pathogens”generally refer to microorganisms, including bacteria, fungi, protozoan,archaea, protists, e.g., algae, and a combination thereof. The term“microbes” also includes pathogenic microbes, e.g., bacteria causingdiseases such as plague, tuberculosis and anthrax; protozoa causingdiseases such as malaria, sleeping sickness and toxoplasmosis; fungicausing diseases such as ringworm, candidiasis or histoplasmosis; andbacteria causing diseases such as sepsis. The term “microbe” or“microbes” can also encompass non-pathogenic microbes, e.g., somemicrobes used in industrial applications.

In some embodiments, the term “microbe” or “microbes” also encompassesfragments of microbes, e.g., cell components of microbes, LPS, and/orendotoxin.

One skilled in the art can understand that the method described hereincan be used to determine the antibiotic susceptibility of anymicroorganism.

In some other embodiments, the method described herein can be used todetermine the antibiotic susceptibility of at least one of the followingpathogens that causes diseases: Bartonella henselae, Borreliaburgdorferi, Campylobacter jejuni, Campylobacterfetus, Chlamydiatrachomatis, Chlamydia pneumoniae, Chylamydia psittaci, Simkanianegevensis, Escherichia coli (e.g., O157:H7 and K88), Ehrlichiachafeensis, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Enterococcus faecalis, Haemophilius influenzae, Haemophiliusducreyi, Coccidioides immitis, Bordetella pertussis, Coxiella burnetii,Ureaplasma urealyticum, Mycoplasma genitalium, Trichomatis vaginalis,Helicobacter pylori, Helicobacter hepaticus, Legionella pneumophila,Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacteriumavium, Mycobacterium celatum, Mycobacterium celonae, Mycobacteriumfortuitum, Mycobacterium genavense, Mycobacterium haemophilum,Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacteriummalmoense, Mycobacterium marinum, Mycobacterium scrofulaceum,Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium ulcerans,Mycobacterium xenopi, Corynebacterium diptheriae, Rhodococcus equi,Rickettsia aeschlimannii, Rickettsia africae, Rickettsia conorii,Arcanobacterium haemolyticum, Bacillus anthracis, Bacillus cereus,Lysteria monocytogenes, Yersinia pestis, Yersinia enterocolitica,Shigella dysenteriae, Neisseria meningitides, Neisseria gonorrhoeae,Streptococcus bovis, Streptococcus hemolyticus, Streptococcus mutans,Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcussaprophyticus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonellatyphi, Salmonella paratyphi, Salmonella enteritidis, Treponema pallidum,Human rhinovirus, Human coronavirus, Dengue virus, Filoviruses (e.g.,Marburg and Ebola viruses), Hantavirus, Rift Valley virus, Hepatitis B,C, and E, Human Immunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8,Human papillomavirus, Herpes virus (e.g., HV-I and HV-II), Human T-celllymphotrophic viruses (e.g., HTLV-I and HTLV-II), Bovine leukemia virus,Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubellavirus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, EpsteinBahr virus, Norwalk (and Norwalk-like) viruses, Rotavirus, ParvovirusB19, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis,Sabia virus, West Nile virus, Yellow Fever virus, causative agents oftransmissible spongiform encephalopathies, Creutzfeldt-Jakob diseaseagent, variant Creutzfeldt-Jakob disease agent, Candida, Cryptcooccus,Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax,Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes,Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoonhellem, Encephalitozoon cuniculi, among other viruses, bacteria,archaea, protozoa, and fungi).

In some embodiments, the method described herein can be used todetermine the antibiotic susceptibility of a bacteria present in abiofilm. For example, Listeria monocytogenes can form biofilms on avariety of materials used in food processing equipment and other foodand non-food contact surfaces (Blackman, J Food Prot 1996; 59:827-31;Frank, J Food Prot 1990; 53:550-4; Krysinski, J Food Prot 1992;55:246-51; Ronner, J Food Prot 1993; 56:750-8). Biofilms can be broadlydefined as microbial cells attached to a surface, and which are embeddedin a matrix of extracellular polymeric substances produced by themicroorganisms. Biofilms are known to occur in many environments andfrequently lead to a wide diversity of undesirable effects. For example,biofilms cause fouling of industrial equipment such as heat exchangers,pipelines, and ship hulls, resulting in reduced heat transfer, energyloss, increased fluid frictional resistance, and accelerated corrosion.Biofilm accumulation on teeth and gums, urinary and intestinal tracts,and implanted medical devices such as catheters and prosthesesfrequently lead to infections (Characklis W G. Biofilm processes. In:Characklis W G and Marshall K C eds. New York: John Wiley & Sons,1990:195-231; Costerton et al., Annu Rev Microbiol 1995; 49:711-45).

In some embodiments, the method described herein can be used todetermine the antibiotic susceptibility of a plant pathogen. Plant fungihave caused major epidemics with huge societal impacts. Examples ofplant fungi include, but are not limited to, Phytophthora infestans,Crinipellis perniciosa, frosty pod (Moniliophthora roreri), oomycetePhytophthora capsici, Mycosphaerella fijiensis, Fusarium Ganoderma sppfungi and Phytophthora. An exemplary plant bacterium includesBurkholderia cepacia. Exemplary plant viruses include, but are notlimited to, soybean mosaic virus, bean pod mottle virus, tobacco ringspot virus, barley yellow dwarf virus, wheat spindle streak virus, soilborn mosaic virus, wheat streak virus in maize, maize dwarf mosaicvirus, maize chlorotic dwarf virus, cucumber mosaic virus, tobaccomosaic virus, alfalfa mosaic virus, potato virus X, potato virus Y,potato leaf roll virus and tomato golden mosaic virus.

In yet other embodiments, the method described herein can be used todetermine the antibiotic susceptibility of bioterror agents (e.g., B.Anthracis, and smallpox).

Embodiments of Various Aspects described herein can be Defined in any ofthe Following Numbered Paragraphs

1. A method of detecting or capturing at least one target entitycomprising:

contacting a sample with a composition comprising a target-binding agentand a blocking agent bound thereto,

wherein the blocking agent is selected for reducing the binding of atleast one interfering agent present in the sample to the target-bindingagent, while permitting a first target entity, if present in the sample,to (a) displace the blocking agent bound to the target-binding agent, orto (b) bind to the target-binding agent without the blocking agent boundthereto.

2. The method of paragraph 1, wherein the blocking agent is selected tohave an effective binding affinity for the target-binding agent that islower than the effective binding affinity of the first target entity forthe target-binding agent; and wherein the blocking agent is selected tohave the effective binding affinity for the target-binding agent that ishigher than the effective binding affinity of at least one interferingagent present in the sample for the target-binding agent.3. The method of paragraph 1 or 2, wherein the effective bindingaffinity of the blocking agent is a function of properties comprisingsurface composition of the blocking agent, avidity, single-bond affinityor affinity, surface composition of the target-binding agent,concentration of the blocking agent, concentration of the first targetentity in the sample, concentration of said at least one interferingagent, or any combinations thereof.4. The method of any of paragraphs 1-3, wherein the effective bindingaffinity of the first target entity is a function of propertiescomprising surface composition of the first target entity, avidity,single-bond affinity or affinity, surface composition of thetarget-binding agent, concentration of the blocking agent, concentrationof the first target entity in the sample, concentration of said at leastone interfering agent, or any combinations thereof.5. The method of any of paragraphs 1-4, wherein the effective bindingaffinity of said at least one interfering agent is a function ofproperties comprising composition of the interfering agent, avidity,single-bond affinity or affinity, surface composition of thetarget-binding agent, concentration of the blocking agent, concentrationof the first target entity in the sample, concentration of said at leastone interfering agent, or any combinations thereof.6. The method of any of paragraphs 1-5, wherein the first target entityis a microbe or a fragment thereof.7. The method of any of paragraphs 1-6, wherein the target-binding agentcomprises a microbe-binding agent.8. The method of paragraph 7, wherein the microbe-binding agentcomprises a lectin (e.g., a FcMBL molecule).

9. The method of any of paragraphs 1-8, wherein the blocking agent is asaccharide.

10. The method of any of paragraphs 1-9, wherein the blocking agent is amonomer, which has no free binding site after binding to thetarget-binding agent.

11. The method of paragraph 10, wherein the monomer is a monosaccharideor modification thereof.

12. The method of any of paragraphs 1-9, wherein the blocking agent is amultimer which has at least one free binding site after binding to thetarget-binding agent.

13. The method of paragraph 12, wherein the multimer is a disaccharide,an oligosaccharide, a polysaccharide, modifications thereof, or anycombinations thereof.14. The method of any of paragraphs 9-13, wherein the saccharide isselected from the group consisting of hexose (e.g., glucose), mannose,maltose, N-acetyl-muramic acid, amino sugars (e.g., galactosamine,glucosamine, sialic acid, N-acetylgludosamine), sulfosugars (e.g.,sulfoquinovose), trehalose, cellobiose, lactose, lactulose, sucrose,fructo-oligosaccharides, cellulose, chitin, or any combinations thereof.15. The method of paragraph 14, wherein the saccharide is selected fromthe group consisting of glucose, maltose, N-acetyl-muramic acid, or anycombinations thereof.16. The method of any of paragraphs 1-15, wherein the blocking agentcomprises glucose.17. The method of any of paragraphs 1-16, wherein said at least oneinterfering agent is a blood cell and/or a fragment thereof present inthe sample, e.g., a red blood cell (or an erythryocyte) and/or afragment thereof.18. The method of any of paragraphs 1-16, wherein said at least oneinterfering agent is a second target entity to be captured or detected.19. The method of any of paragraphs 1-16, wherein said at least oneinterfering agent is a non-specific binding molecule, or a specific butlower affinity binding molecule.20. The method of any of paragraphs 1-19, wherein the effective bindingaffinity of the blocking agent for the target-binding agent is lowerthan the effective binding affinity of the first target entity for thetarget-binding agent by at least about 10%.21. The method of any of paragraphs 1-20, wherein the effective bindingaffinity of the blocking agent for the target-binding agent is higherthan the effective binding affinity of at least one interfering agentpresent in the sample for the target-binding agent by at least about10%.22. The method of any of paragraphs 1-21, wherein the effective bindingaffinity of the blocking agent for the target-binding agent is selectedfor increasing specificity of the target-binding agent to the firsttarget entity in the sample, as compared to the specificity in theabsence of the blocking agent.23. The method of any of paragraphs 1-22, wherein the effective bindingaffinity of the blocking agent for the target-binding agent, asindicated by a dissociation constant for the binding of the blockingagent to the target-binding agent, ranges from about 1 nM to about 500mM, or about 1 μM to about 100 mM, or about 1 mM to about 50 mM.24. The method of any of paragraphs 1-23, wherein the effective bindingaffinity of the first target entity for the target-binding agent, asindicated by a dissociation constant for the binding of the first targetentity to the target-binding agent, is less than 25 mM, less than 1 mM,less than 1 μM, or less than 1 nM.25. The method of any of paragraphs 1-24, wherein the effective bindingaffinity of said at least one interfering agent for the target-bindingagent, as indicated by a dissociation constant for the binding of theinterfering agent to the target-binding agent, is more than 500 μM, ormore than 1 mM, or more than 10 mM.26. The method of any of paragraphs 1-25, wherein the blocking agent ispresent in a pre-determined concentration that does not reduce thebinding of the first target entity to the target-binding agent by morethan 30%, as compared to the binding in the absence of the blockingagent.27. The method of any of paragraphs 1-26, wherein the pre-determinedconcentration of the blocking agent is sufficient to not decreasedetection sensitivity of the target-binding agent binding to the firsttarget entity in the sample by at least about 10%, when compared to thedetection sensitivity in the absence of the blocking agent.28. The method of any of paragraphs 1-27, further comprising exposingthe target-binding agent to the blocking agent at the pre-determinedconcentration to form the composition comprising the target-bindingagent and the blocking agent bound thereto.29. The method of any of paragraphs 1-28, further comprising acompetitive washing to release said at least one interfering agent thatis bound to the target-binding agent after the contacting.30. The method of any of paragraphs 1-29, further comprising separatingthe target-binding agent from the sample after the contacting.31. The method of any of paragraphs 1-30, further comprising detectingthe displaced blocking agent.32. The method of any of paragraphs 1-31, wherein the blocking agentcomprises a detectable label.33. The method of any of paragraphs 1-32, further comprising detectingthe first target entity that is bound to the target-binding agent.34. The method of paragraph 33, wherein the first target entity that isbound to the target-binding agent is detected by a method comprisingcontacting the bound first target entity with a detection agent.35. The method of any of paragraphs 1-34, wherein the sample is selectedfrom the group consisting of a biological sample (e.g., bodily fluidssuch as blood, cells, tissue samples), an environmental sample, a cellculture sample, a blood culture, water, pharmaceutical preparations,foods, beverages, and any combinations thereof.36. The method of any of paragraphs 1-35, wherein the sample is a fluidsample.37. The method of paragraph 36, wherein the fluid sample comprises bloodor serum.38. The method of any of paragraphs 1-37, wherein the sample comprisesor is attached to a solid substrate.39. The method of any of paragraphs 1-38, wherein the compositioncomprises a solid substrate affixed with the target-binding agent.40. The method of paragraph 38 or 39, wherein the solid substrate isselected from the group consisting of a nucleic acid scaffold, a proteinscaffold, a lipid scaffold, a dendrimer, microparticle or a microbead, ananotube, a microtiter plate, a medical apparatus or implant, amicrochip, a filtration device, a membrane, a diagnostic strip, adipstick, an extracorporeal device, a mixing element (e.g., a spiralmixer), a microscopic slide, a hollow fiber, a hollow fiber cartridgeand any combinations thereof.41. The method of any of paragraphs 1-40, wherein the target-bindingagent, the blocking agent and the first target entity are eachindependently selected from the group consisting of cells, peptides,polypeptides, proteins, peptidomimetics, antibodies, antibody fragments(e.g., antigen binding fragments of antibodies), carbohydrate-bindingprotein, e.g., lectins, glycoproteins, glycoprotein-binding molecules,amino acids, carbohydrates (including mono-, di-, tri- andpoly-saccharides), lipids, steroids, hormones, lipid-binding molecules,cofactors, nucleosides, nucleotides, nucleic acids (e.g., DNA or RNA,analogues and derivatives of nucleic acids, or aptamers), peptideaptamers, peptidoglycan, lipopolysaccharide, small molecules, endotoxins(e.g., bacterial lipopolysaccharide), and any combinations thereof.42. The method of paragraph 41, wherein the cells are selected from thegroup consisting of prokaryotes (e.g., microbes such as bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, fungi), bloodcells, and any fragments thereof.43. A composition comprising a target-binding agent and at least oneblocking agent at a pre-determined concentration, wherein the effectivebinding affinity of said at least one blocking agent for thetarget-binding agent is lower than the effective binding affinity of atarget entity to be captured, and wherein the effective binding affinityof said at least one blocking agent for the target-binding agent ishigher than the effective binding affinity of at least one interferingmolecule present in a sample to be assayed for the target-binding agent.44. The composition of paragraph 43, said at least one blocking agent ispre-bound to the target-binding agent.45. The composition of paragraph 43 or 44, wherein the target-bindingagent and said at least one blocking agent are present in a bufferedsolution.46. The composition of any of paragraphs 43-45, further comprising asolid substrate affixed with the target-binding agent.47. The composition of paragraph 46, wherein the solid substrate isselected from the group consisting of a nucleic acid scaffold, a proteinscaffold, a lipid scaffold, a dendrimer, microparticle or a microbead, ananotube, a microtiter plate, a medical apparatus or implant, amicrochip, a filtration device, a membrane, a diagnostic strip, adipstick, an extracorporeal device, a mixing element (e.g., a spiralmixer), a microscopic slide, a hollow fiber, a hollow fiber cartridge,and any combinations thereof.48. The composition of any of paragraphs 43-47, wherein said at leastone interfering agent is a second target molecule to be captured ordetected.49. The composition of any of paragraphs 43-48, wherein said at leastone interfering agent is a non-specific binding molecule, or a specificbut lower affinity binding molecule.50. The composition of any of paragraphs 43-49, wherein thetarget-binding agent, the blocking agent, and said at least oneinterfering agent are each independently selected from the groupconsisting of peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptide aptamers, peptidoglycan,lipopolysaccharide, small molecules, endotoxins (e.g., bacteriallipopolysaccharide), cells, and any combinations thereof.51. The composition of any of paragraphs 43-50, wherein thetarget-binding agent comprises an antibody.52. The composition of any of paragraphs 43-50, wherein thetarget-binding agent comprises a microbe-binding agent.53. The composition of paragraph 52, wherein the microbe-binding agentcomprises a lectin (e.g., a FcMBL molecule).54. The composition of any of paragraphs 43-53, wherein said at leastone blocking agent comprises glucose, maltose, N-acetyl-muramic acid, orany combinations thereof.55. The composition of any of paragraphs 43-54, wherein said at leastone blocking agent comprises glucose.56. The composition of paragraph 55, wherein the pre-determinedconcentration of glucose ranges from about 5 mM to about 200 mM.57. The composition of any of paragraphs 43-56, wherein said at leastone blocking agent comprises a detectable label.58. A kit (for multiplexing) comprising:

a first composition comprising a first target-binding agent and at leastone first blocking agent at a first pre-determined concentration,wherein the effective binding affinity of said at least one firstblocking agent for the first target-binding agent is lower than theeffective binding affinity of a first target entity to be captured, andwherein the effective binding affinity of said at least one firstblocking agent for the first target-binding agent is higher than theeffective binding affinity of at least one first interfering moleculepresent in a sample to be assayed for the first target-binding agent;and

instructions for using the composition for detecting or capturing thefirst target entity.

59. The kit of paragraph 58, further comprising:

a second composition comprising a second target-binding agent andoptionally at least one second blocking agent at a second pre-determinedconcentration, wherein the effective binding affinity of said at leastone second blocking agent for the second target-binding agent is lowerthan the effective binding affinity of a second target entity to becaptured, and wherein the effective binding affinity of said at leastone second blocking agent for the second target-binding agent is higherthan the effective binding affinity of at least one second interferingmolecule present in the sample to be assayed for the secondtarget-binding agent.

60. The kit of paragraph 58 or 59, wherein said at least the firstinterfering agent and said at least the second interfering agent are thesame.61. The kit of any of paragraphs 59-60, wherein said at least the firstinterfering agent and said at least the second interfering agent aredifferent.62. The kit of any of paragraphs 58-60, wherein said at least the firstinterfering agent comprises the second target entity and/or a thirdtarget entity.63. The kit of any of paragraphs 59-60, wherein said at least the secondinterfering agent comprises the first target entity and/or the thirdtarget entity.64. The kit of any of paragraphs 59-60, wherein said at least the firstinterfering agent and/or said at least the second interfering agent is anon-specific binding molecule.65. The kit of any of paragraphs 58-64, wherein the first target-bindingagent is affixed to a first solid substrate.66. The kit of paragraph 65, wherein the first solid substrate isfurther affixed with the second target-binding agent.67. The kit of any of paragraphs 59-66, wherein the secondtarget-binding agent is affixed to a second solid substrate.68. The kit of any of paragraphs 58-67, wherein the first or the secondsolid substrate is selected from the group consisting of a nucleic acidscaffold, a protein scaffold, a lipid scaffold, a dendrimer,microparticle or a microbead, a nanotube, a microtiter plate, a medicalapparatus or implant, a microchip, a filtration device, a membrane, adiagnostic strip, a dipstick, an extracorporeal device, a mixing element(e.g., a spiral mixer), a microscopic slide, a hollow fiber, a hollowfiber cartridge, and any combination thereof.69. The kit of any of paragraphs 58-68, further comprising a firstdetection agent capable of binding to the first target entity.70. The kit of any of paragraphs 59-69, further comprising a seconddetection agent capable of binding to the second target entity.71. A method of detecting or capturing at least one target entitycomprising:

contacting a sample with a composition comprising a target-binding agentand a blocking agent bound thereto, wherein the blocking agent isselected for reducing the binding of at least one interfering agentpresent in the sample to the target-binding agent, while permitting afirst target entity, if present in the sample, to (a) displace theblocking agent bound to the target-binding agent, or to (b) bind to thetarget-binding agent without the blocking agent bound thereto.

72. The method of paragraph 71, wherein the blocking agent is selectedto have an effective binding affinity for the target-binding agent thatis lower than the effective binding affinity of the first target entityfor the target-binding agent; and wherein the blocking agent is selectedto have the effective binding affinity for the target-binding agent thatis higher than the effective binding affinity of at least oneinterfering agent present in the sample for the target-binding agent.73. The method of 71 or 72, wherein the first target entity is a microbeor a fragment thereof and the target-binding agent comprises amicrobe-binding agent.74. The method of paragraph 73, wherein the microbe-binding agentcomprises a carbohydrate recognition domain derived from at least onecarbohydrate-binding protein selected from the group consisting oflectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, glucose-bindingprotein, Galanthus nivalis agglutinin, peanut lectin, lentil lectin,DC-SIGN, C-reactive protein, and any combinations thereof.75. The method of paragraph 74, wherein the microbe-binding agentcomprises an amino acid sequence selected from SEQ ID NO: 1-SEQ ID NO:8.76. The method of any of paragraphs 71-75, wherein the blocking agent isa monomer, the monomer having no free binding site after binding to thetarget-binding agent; or a multimer, the multimer having at least onefree-binding site after binding to the target-binding agent.77. The method of any of paragraphs 71-76, wherein the blocking agent isa saccharide.78. The method of paragraph 77, wherein the saccharide is selected fromthe group consisting of hexose, glucose, mannose, maltose,N-acetyl-muramic acid, amino sugars (e.g., galactosamine, glucosamine,sialic acid, N-acetylgludosamine), sulfosugars (e.g., sulfoquinovose),trehalose, cellobiose, lactose, lactulose, sucrose,fructo-oligosaccharides, cellulose, chitin, or any combinations thereof.79. The method of any of paragraphs 71-78, wherein said at least oneinterfering agent is selected from the group consisting of

a. a blood cell and/or a fragment thereof present in the sample, e.g., ared blood cell (or an erythrocyte) and/or a fragment thereof;

b. a second target entity to be captured or detected;

c. a non-specific binding molecule;

d. a specific but low affinity binding molecule; and

e. any combinations thereof.

80. The method of any of paragraphs 71-79, wherein the effective bindingaffinity of the blocking agent for the target-binding agent is measuredby a dissociation constant, wherein the dissociation constant forbinding of the blocking agent to the target-binding agent ranges fromabout 1 mM to about 50 mM.81. The method of any of paragraphs 71-80, further comprising, prior tothe contacting, exposing the target-binding agent to the blocking agentto form the composition comprising the target-binding agent and theblocking agent bound thereto.82. The method of any of paragraphs 71-81, further comprising acompetitive washing to release said at least one interfering agent thatis bound to the target-binding agent after the contacting.83. The method of any of paragraphs 71-82, further comprising separatingthe target-binding agent from the sample after the contacting.84. The method of any of paragraphs 71-83, further comprising detectingthe at least one target entity that is bound to the target-bindingagent.85. The method of paragraph 84, wherein the at least one target entityis detected by a method comprising contacting the bound target entitywith a detection agent, wherein the detection agent does not bind to theblocking agent.86. The method of any of paragraphs 71-85, wherein the sample isselected from the group consisting of a biological sample (e.g., bodilyfluids such as blood, cells, tissue samples), an environmental sample, acell culture sample, a blood culture, water, pharmaceuticalpreparations, foods, beverages, solid supports (e.g., membranes, slides,plates) comprising the at least one target entity, and any combinationsthereof.87. The method of paragraph 86, wherein the sample is a fluid samplecomprising blood or serum.88. The method of any of paragraphs 71-87, wherein the target-bindingagent is attached to a solid substrate.89. The method of paragraph 88, wherein the solid substrate is selectedfrom the group consisting of a nucleic acid scaffold, a proteinscaffold, a lipid scaffold, a dendrimer, microparticle or a microbead, ananotube, a microtiter plate, a medical apparatus or implant, amicrochip, a filtration device, a membrane, a diagnostic strip, adipstick, an extracorporeal device, a mixing element (e.g., a spiralmixer), a microscopic slide, a hollow fiber, a hollow fiber cartridge,and any combinations thereof.90. A composition comprising a target-binding agent and a blockingagent, wherein the effective binding affinity of the blocking agent forthe target-binding agent is lower than the effective binding affinity ofa target entity to be captured, and wherein the effective bindingaffinity of the blocking agent for the target-binding agent is higherthan the effective binding affinity of at least one interfering moleculepresent in a sample to be assayed for the target-binding agent.91. The composition of paragraph 90, wherein the target-binding agent isa microbe-binding agent comprising a carbohydrate recognition domainderived from at least one carbohydrate-binding protein selected from thegroup consisting of lectin, collectin, ficolin, mannose-binding lectin(MBL), maltose-binding protein, arabinose-binding protein,glucose-binding protein, Galanthus nivalis agglutinin, peanut lectin,lentil lectin, DC-SIGN, C-reactive protein, and any combinationsthereof.92. The composition of paragraph 91, wherein the microbe-binding agentfurther comprises (i) a linker (e.g., Fc portion) linked to carbohydraterecognition domain; and optionally (ii) a substrate binding domainadapted for orienting the carbohydrate recognition domain away from asolid substrate surface.93. The composition of paragraph 92, wherein the solid substrate isselected from the group consisting of a nucleic acid scaffold, a proteinscaffold, a lipid scaffold, a dendrimer, microparticle or a microbead, ananotube, a microtiter plate, a medical apparatus or implant, amicrochip, a filtration device, a membrane, a diagnostic strip, adipstick, an extracorporeal device, a mixing element (e.g., a spiralmixer), a microscopic slide, a hollow fiber, a hollow fiber cartridge,and any combinations thereof.94. The composition of any of paragraphs 90-93, said at least oneblocking agent is pre-bound to the target-binding agent.95. The composition of any of paragraphs 90-94, wherein said at leastone blocking agent comprises a saccharide selected from the groupconsisting of hexose, glucose, mannose, maltose, N-acetyl-muramic acid,amino sugars (e.g., galactosamine, glucosamine, sialic acid,N-acetylgludosamine), sulfosugars (e.g., sulfoquinovose), trehalose,cellobiose, lactose, lactulose, sucrose, fructo-oligosaccharides,cellulose, chitin, and any combinations thereof.96. A kit for differentiating a first target entity from a second targetentity in a sample comprising:

a first composition comprising a first target-binding agent and ablocking agent, wherein the effective binding affinity of the blockingagent for the first target-binding agent is lower than the effectivebinding affinity of the first target entity for the first target-bindingagent, and wherein the effective binding affinity of the blocking agentfor the first target-binding agent is higher than the effective bindingaffinity of the second target entity for the first target-binding agent;

a second composition comprising a second target-binding agent; and

instructions for using the composition for differentiating a firsttarget entity from a second target entity in a sample.

Some Selected Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means ±1%.

In one aspect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these

As used herein, the term “peptidomimetic” refers to a molecule capableof folding into a defined three-dimensional structure similar to anatural peptide.

As used herein, the term “small molecules” refers to natural orsynthetic molecules including, but not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,organic or inorganic compounds (i.e., including heteroorganic andorganometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1. Improvement of Lectin Binding Specificity using Competing LowAffinity Substrates (Blocking Agent)

Lectins are proteins that have binding sites for specific mono- oroligo-saccharides. Lectins recognize a range of different carbohydratesand carbohydrate-containing patterns based on a number of factors,including, e.g., the chemical nature of the carbohydrate, and/or thedensity or geometry of the carbohydrate relative to its tethering point.Different lectins (either native or engineered) have differentaffinities towards different ligands. The low affinity binding oflectins to some carbohydrate moieties limit the usefulness of lectins inmany biological applications.

Accordingly, in one embodiment, presented herein is a method to improvethe binding specificity of a target-binding agent comprising lectin inan assay by the addition of a blocking agent, e.g., a binding substrateof intermediate affinity, in a competitive manner. An example of abinding substrate of intermediate affinity is a binding substrate withan effective binding affinity for the target-binding agent between theeffective binding affinity of a target entity (e.g., a microbe or afragment thereof) for the target-binding agent and the effective bindingaffinity of an interfering agent present in a sample (e.g., a red bloodcell or a fragment thereof) for the target-binding agent.

By way of example only, assuming that substance A is the preferredligand (target entity) of a given target binding agent (e.g., lectin L),L can be used to bind A for any purpose, such as purification,detection, removal or other. However, L can also bind to a substance Bwith lower affinity (an interfering agent) which will impair thepurification, detection, and/or removal of A by L. In a complex matrixsuch as blood or serum or food or other mixtures, it is often found thatL binds to the more abundant B therefore decreasing the performance of Lbinding to A. Accordingly, some embodiments of the methods describedherein relates to adding a blocking agent (e.g., carbohydrate C ofintermediate affinity to L), which is preferentially bound over B buteasily displaced by A, in order to improve binding specificity and/orsensitivity of L to A. The adequate choice of C is selected such that itdoes not interfere with the desired function assigned to L, e.g., Cbinding to L does not compete with the binding of A to L but preventsthe binding of B to L.

In one embodiment where L is FcMBL, A is mannan, B is glucose, it isfound that in a simple controlled medium where mannan is the solecarbohydrate present, FcMBL can detect mannan down to amounts equal orlower than 2 ng/ml. However, when mannan is added to a complex mediumsuch as blood, the detection of mannan is impaired by a background,e.g., the binding of erythrocytes or other carbohydrates via the mannosebinding domains on FcMBL. The binding of erythrocytes (or othercarbohydrates) can impair downstream detection because bounderythrocytes generally interfere with the successive detection steps,thereby raising the detection threshold (up to 500 ng/ml or more from 2ng/ml).

The inventors have discovered that addition of a small amount of mannancan decrease the background by preventing the binding of erythrocytes orother endogenous components from blood to FcMBL.

The inventors have also discovered that the addition of a mediumaffinity ligand of FcMBL such as glucose (K_(d)=23 mM), or maltose(K_(d)=15 mM) or N-acetyl Muramic acid (MurNAc, K_(d)=17 mM) to FcMBLprior to the capture of mannan in the presence of blood decreases thebackground of the FcMBL ELISA. Without wishing to be bound by theory,this reduced background can be related to the prevention of FcMBL lowaffinity binding to erythrocytes (or other endogenous components inblood) by the medium affinity sugar that in turn can be displaced by thehigher affinity targets such as mannan, lipopolysaccharides (LPS),and/or a microbe or fragment thereof.

As shown in FIG. 1, the addition of ˜10 mM glucose does not adverselyaffect mannan binding in buffer, whereas ˜20 mM glucose effectivelyreduces the mannan binding by ˜50% and ˜40 mM glucose almost abolishesthe mannan binding in buffer. Further, the addition of ˜10 mM glucosedecreases non-specific binding of FcMBL to blood without affecting thedetection of mannan in donor blood. Accordingly, the concentration of ablocking agent (e.g., glucose) for use in an assay (e.g., FcMBL assay)is selected such that the presence of the blocking agent (e.g., glucose)does not significantly affect target binding (e.g., mannan binding inthis Example), while sufficient to prevent the binding of interferingagents (e.g., erythrocytes) to a target-binding agent (e.g., FcMBL).

FcMBL also has a high affinity for bacterial lipopolysaccharide (LPS)also known as endotoxin. As shown in FIG. 2, competition assays showthat higher concentrations of glucose are required to effectivelycompete for binding to FcMBL. Unlike mannan detection, the addition of˜40 mM glucose or ˜80 mM glucose does not significantly affect thedetection of LPS in serum. When glucose was added at a concentration ofabout 160 mM, the LPS detection was reduced to 10% as compared to thedetection level determined in the absence of glucose.

FIG. 3A shows that the addition of glucose reduces the background noisecontributed by interfering agents present in donor blood, therebyincreasing the specificity of FcMBL binding to LPS, as evidenced bydecreasing OD₄₅₀ signal as the concentration of LPS spiked in donorblood decreases. Further, FIG. 3B shows that the addition of glucosesignificantly decreases the binding of haemocytes (e.g., erythrocytes)to FcMBL in donor blood.

In addition to reduced background, the presence of a medium affinitytarget (e.g., but not limited to glucose) for capture can be used toreduce false positives in the assays resulting from captured non-targetmaterials such as erythrocytes. For example, as shown in FIG. 4, theFcMBL ELISA assay indicates that a microbe is detected in a clinicalsample of patient 1109, when the assay was performed without addition ofa blocking agent (e.g., glucose). However, the positive microbialdetection disappeared when the assay was performed in the presence of ablocking agent (e.g., glucose) at an appropriate concentration (e.g.,˜10 mM). The addition of glucose can prevent binding of non-targetmaterials such as erythrocytes from binding to FcMBL, which wouldotherwise contribute to a false-positive.

In some embodiments, the competition of a blocking agent (e.g., glucose)over an interfering agent (e.g., erythrocytes) can also permit theinclusion of at least one competitive wash step to release theinterfering agent (e.g., erythrocytes) that are still bound to thetarget-binding agent (e.g., FcMBL), thereby increasing thestringency/sensitivity of the detection reaction. For example, aftercontacting a sample with a composition comprising a target-binding agent(e.g., FcMBL) and a blocking agent (e.g., glucose) bound to thetarget-binding agent (e.g., FcMBL), the mixture can be washed with awash buffer comprising a blocking agent (e.g., glucose) so as to removeany residual interfering agent (e.g., erythrocytes) not competed away bythe target-binding agent (e.g., FcMBL), e.g., due to low abundance ofthe target-binding agent (e.g., FcMBL).

The methods described herein comprising addition of a blocking agent toan assay are applicable to all functions performed by FcMBL in additionto microbial detection. In the use of FcMBL for depletion of microbesfrom blood or food, e.g., for detection purposes, glucose can be addedto a storage buffer and prebound to FcMBL. The high affinity binding ofthe captured microorganism can displace the blocking agent (e.g., sugarsuch as glucose) and prevent undesirable binding of blood cells etc.,that would interfere with downstream detection processes, e.g.,ATP-based detection of viable bacteria or downstream geneticamplification efficiency or immunoenzymatic detection. While performingan assay using a dialysis-like therapeutic (DLT) device, e.g., asdescribed in the International Application Publication No. WO2012/135834, the content of which is incorporated herein by reference,FcMBL beads or membrane can be preloaded with a blocking agent (e.g.,glucose or maltose etc.) to not only enhance the capture of pathogensand microbial carbohydrate compounds but also to enhance magnetic beadrecovery and prevent FcMBL inactivation by low affinity binders such aserythrocytes.

The addition of a blocking agent to a test sample in a competitivemanner can be applied to any other protein-ligand system where lowaffinity binders interfere. For example, in an antibody (Ab)-basedassay, some common interfering epitopes are low affinity binders and cancompromise the assay.

In one embodiment, the addition of a blocking agent to a test sample ina competitive manner can be used in immunoglobulin secondary detectionreactions. An example of such an application is described below:Fluorescent-labeled IgG1 has been raised to detect rabbit F(c) fragmentfor which IgG1 has high affinity. However, IgG1 also has a low affinityto goat F(c) and a medium affinity to an aptamer derived from the rabbitF(c) epitope. Thus, incubating HRP-labeled IgG1 in multiplex labelingassay where a goat primary Ab and a rabbit primary Ab are both used canresult in the fluorescent labeling of both the goat and rabbit primaryAbs.

In order to distinguish the labeling of both the goat and rabbit primaryAbs, fluorescent-labeled IgG1 can be incubated with the aptamers(derived from the rabbit F(c) epitope) with a medium affinity prior toaddition into a multiplex labeling assay where both a goat primary Aband a rabbit are used. In this example, the high affinity ligand A isthe rabbit Ab, the low affinity undesirable ligand B is the goat Ab andthe intermediate affinity ligand C is the aptamer. The rabbit Ab (A) candisplace the aptamers (C) that are bound to the fluorescent-labeled IgG1but the goat Ab (B) is less likely to bind to the fluorescent-labeledIgG1 because the fluorescent-labeled IgG1 has already bound to theaptamers, which cannot be displaced by the goat Ab (B) with loweraffinity. In some embodiments, the goat Ab (B) that is not bound to thefirst fluorescent-labeled IgG1 can be then detected with anotherfluorescent-labeled IgG1, thus enabling detection of different targetentities in a multiplex labeling assay, e.g., using the same detectionagent (e.g., IgG1) but with a different detectable label (e.g., adifferent fluorescent label) for each target entity (e.g., rabbit Ab andgoat Ab).

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

What is claimed is:
 1. A composition comprising a) a target-bindingagent and b) a blocking agent that is bound to the target-binding agentand is displaceable by a target entity to be captured from a sample;wherein the effective binding affinity of the blocking agent for thetarget-binding agent is lower than the effective binding affinity of thetarget entity; and wherein the effective binding affinity of theblocking agent for the target-binding agent is higher than the effectivebinding affinity of at least one interfering agent present in thesample.
 2. The composition of claim 1, further comprising the targetentity bound to the target-binding agent.
 3. The composition of claim 1,wherein the target-binding agent and the blocking agent are present in abuffered solution.
 4. The composition of claim 1, further comprising asolid substrate affixed with the target-binding agent.
 5. Thecomposition of claim 4, wherein the solid substrate is selected from thegroup consisting of: a nucleic acid scaffold; a protein scaffold; alipid scaffold; a dendrimer; microparticle or a microbead; a nanotube; amicrotiter plate; a medical apparatus or implant; a microchip; afiltration device; a membrane; a diagnostic strip; a dipstick; anextracorporeal device; a mixing element; a spiral mixer; a microscopicslide; a hollow-fiber reactor; and any combinations thereof.
 6. Thecomposition of claim 1, wherein said at least one interfering agent is asecond target molecule to be captured or detected.
 7. The composition ofclaim 1, wherein said at least one interfering agent is a non-specificbinding molecule, or a specific but lower affinity binding molecule. 8.The composition of claim 1, wherein the target-binding agent, theblocking agent, and said at least one interfering agent are eachindependently selected from the group consisting of: peptides;polypeptides; proteins; peptidomimetics; antibodies; antibody fragments;antigen binding fragments of antibodies; carbohydrate-binding protein; alectin; glycoproteins; glycoprotein-binding molecules; amino acids;carbohydrates (including mono-; di-; tri- and poly-saccharides); lipids;steroids; hormones; lipid-binding molecules; cofactors; nucleosides;nucleotides; nucleic acids; DNA; RNA; analogues and derivatives ofnucleic acids; nucleic acid aptamers; peptide aptamers; peptidoglycan;lipopolysaccharide; small molecules; endotoxins; bacteriallipopolysaccharide; cells; and any combinations thereof.
 9. Thecomposition of claim 1, wherein the target-binding agent comprises anantibody.
 10. The composition of claim 1, wherein the target-bindingagent comprises a microbe-binding agent.
 11. The composition of claim10, wherein the microbe-binding agent comprises a carbohydraterecognition domain derived from at least one carbohydrate-bindingprotein selected from the group consisting of: lectin; collectin;ficolin; mannose-binding lectin (MBL); maltose-binding protein;arabinose-binding protein; glucose-binding protein; Galanthus nivalisagglutinin; peanut lectin; lentil lectin; DC-SIGN; C-reactive protein;and any combinations thereof.
 12. The composition of claim 10, whereinthe microbe-binding agent comprises a lectin.
 13. The composition ofclaim 10, wherein the microbe-binding agent comprises a FcMBL molecule.14. The composition of claim 10, wherein the microbe-binding agentcomprises an amino acid sequence selected from SEQ ID NO: 1-SEQ ID NO:8.
 15. The composition of claim 1, wherein the blocking agent is amonomer, the monomer having no free binding site after binding to thetarget-binding agent.
 16. The composition of claim 1, wherein theblocking agent is a multimer, the multimer having at least onefree-binding site after binding to the target-binding agent.
 17. Thecomposition of claim 1, wherein the blocking agent comprises glucose,maltose, N-acetyl-muramic acid, or any combinations thereof.
 18. Thecomposition of claim 1, wherein the blocking agent comprises glucose.19. The composition of claim 18, wherein the glucose is present at aconcentration of from 5 mM to 200 mM.
 20. The composition of claim 1,wherein the blocking agent comprises a detectable label.